Methods of using an equine fc epsilon receptor alpha chain protein

ABSTRACT

The present invention relates to equine Fc epsilon receptor alpha chain nucleic acid molecules, proteins encoded by such nucleic acid molecules, antibodies raised against such proteins, and inhibitors of such proteins. The present invention also includes methods to detect IgE using such proteins and antibodies. Also included in the present invention are therapeutic compositions comprising such proteins, nucleic acid molecules, antibodies and/or inhibitory compounds as well as the use of such therapeutic compositions to mediate Fc epsilon receptor-mediated biological responses.

FIELD OF THE INVENTION

The present invention relates to equine Fc epsilon receptor alpha chainnucleic acid molecules, proteins encoded by such nucleic acid molecules,antibodies raised against such proteins, and inhibitors of suchproteins. The present invention also includes methods to detect IgEusing such proteins and antibodies.

BACKGROUND OF THE INVENTION

Diagnosis of disease and determination of treatment efficacy areimportant tools in medicine. IgE antibody production in an animal can beindicative of disease including, for example, allergy, atopic disease,hyper IgE syndrome, internal parasite infections and B cell neoplasia.In addition, detection of IgE production in an animal following atreatment is indicative of the efficacy of the treatment, such as whenusing treatments intended to disrupt IgE production.

Immunological stimulation can be mediated by IgE antibodies when IgEcomplexes with Fc epsilon receptors. Fc epsilon receptors are found onthe surface of certain cell types, such as mast cells. Mast cells storebiological mediators including histamine, prostaglandins and proteases.Release of these biological mediators is triggered when IgE antibodiescomplex with Fc epsilon receptors on the surface of a cell. Clinicalsymptoms result from the release of the biological mediators into thetissue of an animal.

The discovery of the present invention includes a novel equine Fcepsilon receptor (Fc_(ε)R) alpha chain protein and the use of such aprotein to detect the presence of IgE in a putative IgE-containingcomposition; to identify inhibitors of biological responses mediated byan equine Fc_(ε)R protein; and as a therapeutic compound to prevent ortreat clinical symptoms that result from equine Fc_(ε)R-mediatedbiological responses.

Prior investigators have disclosed the nucleic acid sequence for: thehuman Fc_(ε)R alpha chain (Kochan et al., Nucleic Acids Res. 16:3584,1988; Shimizu et al., Proc. Natl. Acad. Sci. USA 85:1907-1911, 1988; andPang et al., J. Immunol. 151:6166-6174, 1993); the human Fc_(ε)R betachain (Kuster et al., J. Biol. Chem. 267:12782-12787, 1992); the humanFc_(ε)R gamma chain (Kuster et al., J. Biol. Chem. 265:6448-6452, 1990);and the canine Fc_(ε)R alpha chain (GenBank™ accession number D16413).Although the subunits of human Fc_(ε)R have been known as early as 1988,they have never been used to identify an equine Fc_(ε)R. Similarly, eventhough the canine Fc_(ε)R chain has been known since 1993, it has neverbeen used to identify an equine Fc_(ε)R. Moreover, the determination ofhuman and canine Fc epsilon receptor sequences does not indicate,suggest or predict the cloning of a novel Fc_(ε)R gene from a differentspecies, in particular, from an equine species. Previous investigatorshave found a low degree of similarity between rat, mouse and humanFc_(ε)Rα (Ravtech et al., Ann. Rev. Immunol. Vol. 9, pp. 457-492, 1991).Thus, given this low degree of sequence similarity, it would appear only“obvious to try” to obtain an equine Fc_(ε)Rα nucleic acid molecule andprotein.

Thus, products and processes of the present invention are needed in theart that will provide specific detection of IgE, in particular equineIgE, and treatment of Fc epsilon receptor-mediated disease.

SUMMARY OF THE INVENTION

The present invention relates to a novel product and process fordetecting IgE and protecting animals from Fc epsilon receptor-mediatedbiological responses. According to the present invention there areprovided equine Fc_(ε)R proteins and mimetopes thereof; equine Fc_(ε)Rnucleic acid molecules, including those that encode such proteins;antibodies raised against such equine Fc_(ε)R proteins (i.e.,anti-equine Fc_(ε)R antibodies); and other compounds that inhibit theability of equine Fc_(ε)R protein to form a complex with IgE (i.e,inhibitory compounds or inhibitors).

The present invention also includes methods to obtain such proteins,mimetopes, nucleic acid molecules, antibodies and inhibitory compounds.Also included in the present invention are therapeutic compositionscomprising such proteins, mimetopes, nucleic acid molecules, antibodies,and/or inhibitory compounds, as well as use of such therapeuticcompositions to Fc epsilon receptor-mediated biological responses.

One embodiment of the present invention is an isolated nucleic acidmolecule encoding an equine Fc_(ε)R protein. The equine Fc_(ε)R proteinpreferably includes: proteins comprising amino acid sequences SEQ IDNO:2, SEQ ID NO:7 and SEQ ID NO:12; and proteins encoded by allelicvariants of nucleic acid molecules encoding a protein comprising any ofthe amino acid sequences. Particularly preferred equine Fc_(ε)R nucleicacid molecules include: nucleic acid molecules comprising nucleic acidsequences SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:8 and SEQ ID NO:11 and nucleic acid molecules comprisingallelic variants of nucleic acid molecules comprising nucleic acidsequences SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:8 and SEQ ID NO:11.

The present invention also includes an isolated equine Fc_(ε)R protein.A preferred equine Fc_(ε)R protein is encoded by a nucleic acid moleculethat hybridizes under stringent hybridization conditions to a nucleicacid sequence including SEQ ID NO:3, SEQ ID NO:5 and SEQ ID NO:8.Particularly preferred equine Fc_(ε)R proteins include at least one ofthe following amino acid sequences: SEQ ID NO:2, SEQ ID NO:7 and SEQ IDNO:12.

The present invention also relates to recombinant molecules, recombinantviruses and recombinant cells that include equine Fc_(ε)R nucleic acidmolecules of the present invention. Also included are methods to producesuch nucleic acid molecules, recombinant molecules, recombinant virusesand recombinant cells.

The present invention also includes detection methods and kits thatdetect IgE. One embodiment of the present invention is a method todetect IgE comprising: (a) contacting an isolated equine Fc_(ε)Rmolecule with a putative IgE-containing composition under conditionssuitable for formation of a Fc_(ε)R molecule:IgE complex; and (b)determining the presence of IgE by detecting the Fc_(ε)R molecule:IgEcomplex, the presence of the Fc_(ε)R molecule:IgE complex indicating thepresence of IgE. A preferred equine Fc_(ε)R molecule is one in which acarbohydrate group of the equine Fc_(ε)R molecule is conjugated tobiotin.

Another embodiment of the present invention is a method to detect IgEcomprising: (a) contacting a recombinant cell with a putativeIgE-containing composition under conditions suitable for formation of arecombinant cell:IgE complex, in which the recombinant cell comprises anequine Fc_(ε)R molecule; and (b) determining the presence of IgE bydetecting the recombinant cell:IgE complex, the presence of therecombinant cell:IgE complex indicating the presence of IgE. A preferredmethod to detect IgE comprises: (a) immobilizing the Fc_(ε)R molecule ona substrate; (b) contacting the Fc_(ε)R molecule with the putativeIgE-containing composition under conditions suitable for formation of aFc_(ε)R molecule:IgE complex bound to the substrate; (c) removingnon-bound material from the substrate under conditions that retainFc_(ε)R molecule:IgE complex binding to the substrate; and (d) detectingthe presence of the Fc_(ε)R molecule:IgE complex. Another preferredmethod to detect IgE comprises: (a) immobilizing a specific antigen on asubstrate; (b) contacting the antigen with the putative IgE-containingcomposition under conditions suitable for formation of an antigen:IgEcomplex bound to the substrate; (c) removing non-bound material from thesubstrate under conditions that retain antigen:IgE complex binding tosaid substrate; and (d) detecting the presence of the antigen:IgEcomplex by contacting the antigen:IgE complex with said Fc_(ε)Rmolecule. Another preferred method to detect IgE comprises: (a)immobilizing an antibody that binds selectively to IgE on a substrate;(b) contacting the antibody with the putative IgE-containing compositionunder conditions suitable for formation of an antibody:IgE complex boundto the substrate; (c) removing non-bound material from the substrateunder conditions that retain antibody:IgE complex binding to thesubstrate; and (d) detecting the presence of the antibody:IgE complex bycontacting the antibody:IgE complex with said Fc_(ε)R molecule. Anotherpreferred method to detect IgE comprises: (a) immobilizing a putativeIgE-containing composition on a substrate; (b) contacting thecomposition with the Fc_(ε)R molecule under conditions suitable forformation of a Fc_(ε)R molecule:IgE complex bound to the substrate; (c)removing non-bound material from the substrate under conditions thatretain Fc_(ε)R molecule:IgE complex binding to the substrate; and (d)detecting the presence of the Fc_(ε)R molecule:IgE complex.

The present invention also includes a kit for performing methods of thepresent invention. One embodiment is a kit for detecting IgE comprisingan equine Fc_(ε)R protein and a means for detecting IgE.

The present invention also includes an inhibitor that interferes withformation of a complex between equine Fc_(ε)R protein and IgE, in whichthe inhibitor is identified by its ability to interfere with the complexformation. A particularly preferred inhibitor includes a substrateanalog of an equine Fc_(ε)R protein, a mimetope of an equine Fc_(ε)Rprotein and a soluble portion of an equine Fc_(ε)R protein. Alsoincluded is a method to identify a compound that interferes withformation of a complex between equine Fc_(ε)R protein and IgE, themethod comprising: (a) contacting an isolated equine Fc_(ε)R proteinwith a putative inhibitory compound under conditions in which, in theabsence of the compound, the equine Fc_(ε)R protein forms a complex withIgE; and (b) determining if the putative inhibitory compound inhibitsthe complex formation. A test kit is also included to identify acompound capable of interfering with formation of a complex between anequine Fc_(ε)R protein and IgE, the test kit comprising an isolatedequine Fc_(ε)R protein that can complex with IgE and a means fordetermining the extent of interference of the complex formation in thepresence of a putative inhibitory compound.

Yet another embodiment of the present invention is a therapeuticcomposition that is capable of reducing Fc epsilon receptor-mediatedbiological responses. Such a therapeutic composition includes one ormore of the following therapeutic compounds: an isolated equine Fc_(ε)Rprotein; a mimetope of an equine Fc_(ε)R protein; an isolated nucleicacid molecule that hybridizes under stringent hybridization conditionswith an equine Fc_(ε)R gene; an isolated antibody that selectively bindsto an equine Fc_(ε)R protein; and an inhibitor that interferes withformation of a complex between an equine Fc_(ε)R protein and IgE. Amethod of the present invention includes the step of administering to ananimal a therapeutic composition of the present invention.

Yet another embodiment of the present invention is a method to producean equine Fc_(ε)R protein, the method comprising culturing a celltransformed with a nucleic acid molecule encoding an equine Fc_(ε)Rprotein.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides for isolated equine Fc epsilon receptoralpha chain (Fc_(ε)Rα) proteins, isolated equine Fc_(ε)Rα nucleic acidmolecules, antibodies directed against equine Fc_(ε)Rα proteins andother inhibitors of equine Fc_(ε)Rα activity. As used herein, the termsisolated equine Fc_(ε)Rα proteins and isolated equine Fc_(ε)Rα nucleicacid molecules refers to Fc_(ε)Rα proteins and Fc_(ε)Rα nucleic acidmolecules derived from horses and, as such, can be obtained from theirnatural source or can be produced using, for example, recombinantnucleic acid technology or chemical synthesis. Also included in thepresent invention is the use of these proteins and antibodies in amethod to detect epsilon immunoglobulin (referred to herein as IgE orIgE antibody) as well as in other applications, such as those disclosedbelow. The products and processes of the present invention areadvantageous because they enable the detection of IgE and the inhibitionof IgE or equine Fc_(ε)Rα protein activity associated with disease. Asused herein, equine Fc epsilon alpha chain receptor protein can bereferred to as Fc_(ε)Rα protein or Fc_(ε)R alpha chain protein.

One embodiment of the present invention is an isolated proteincomprising an equine Fc_(ε)Rα protein. It is to be noted that the term“a” or “an” entity refers to one or more of that entity; for example, aprotein refers to one or more proteins or at least one protein. As such,the terms “a” (or “an”), “one or more” and “at least one” can be usedinterchangeably herein. It is also to be noted that the terms“comprising”, “including”, and “having” can be used interchangeably.Furthermore, a compound “selected from the group consisting of” refersto one or more of the compounds in the list that follows, includingmixtures (i.e., combinations) of two or more of the compounds. Accordingto the present invention, an isolated, or biologically pure, protein, isa protein that has been removed from its natural milieu. As such,“isolated” and “biologically pure” do not necessarily reflect the extentto which the protein has been purified. An isolated protein of thepresent invention can be obtained from its natural source, can beproduced using recombinant DNA technology or can be produced by chemicalsynthesis.

As used herein, an isolated equine Fc_(ε)Rα protein can be a full-lengthprotein or any homolog of such a protein. As used herein, a protein canbe a polypeptide or a peptide. Preferably, an equine Fc_(ε)Rα proteincomprises at least a portion of an equine Fc_(ε)Rα protein that binds toIgE, i.e., that is capable of forming a complex with an IgE.

An equine Fc_(ε)Rα protein of the present invention, including ahomolog, can be identified in a straight-forward manner by the protein'sability to bind to IgE. Examples of equine Fc_(ε)Rα protein homologsinclude equine Fc_(ε)Rα proteins in which amino acids have been deleted(e.g., a truncated version of the protein, such as a peptide), inserted,inverted, substituted and/or derivatized (e.g., by glycosylation,phosphorylation, acetylation, myristoylation, prenylation,palmitoylation, amidation and/or addition of glycerophosphatidylinositol) such that the homolog is capable of binding to IgE.

Equine Fc_(ε)Rα protein homologs can be the result of natural allelicvariation or natural mutation. Equine Fc_(ε)Rα protein homologs of thepresent invention can also be produced using techniques known in the artincluding, but not limited to, direct modifications to the protein ormodifications to the gene encoding the protein using, for example,classic or recombinant nucleic acid techniques to effect random ortargeted mutagenesis.

Isolated equine Fc_(ε)Rα proteins of the present invention have thefurther characteristic of being encoded by nucleic acid molecules thathybridize under stringent hybridization conditions to a gene encoding anequine Fc_(ε)Rα protein. As used herein, stringent hybridizationconditions refer to standard hybridization conditions under whichnucleic acid molecules, including oligonucleotides, are used to identifysimilar nucleic acid molecules. Such standard conditions are disclosed,for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Labs Press, 1989; Sambrook et al., ibid., isincorporated by reference herein in its entirety. Stringenthybridization conditions typically permit isolation of nucleic acidmolecules having at least about 70% nucleic acid sequence identity withthe nucleic acid molecule being used to probe in the hybridizationreaction. Formulae to calculate the appropriate hybridization and washconditions to achieve hybridization permitting 30% or less mismatch ofnucleotides are disclosed, for example, in Meinkoth et al., 1984, Anal.Biochem. 138, 267-284; Meinkoth et al., ibid., is incorporated byreference herein in its entirety.

As used herein, an equine Fc_(ε)Roc gene includes all nucleic acidsequences related to a natural equine Fc_(ε)Rα gene such as regulatoryregions that control production of the equine Fc_(ε)Rα protein encodedby that gene (such as, but not limited to, transcription, translation orpost-translation control regions) as well as the coding region itself.In one embodiment, an equine Fc_(ε)Rα gene of the present inventionincludes nucleic acid sequence SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4,SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8 and/or SEQ ID NO:11. Nucleic acidsequence SEQ ID NO:1 represents the deduced sequence of the codingstrand of a complementary DNA (cDNA) nucleic acid molecule denotedherein as neqFc_(ε)Rα₁₀₁₅, the production of which is disclosed in theExamples. The complement of SEQ ID NO:1 (represented herein by SEQ IDNO:3) refers to the nucleic acid sequence of the strand complementary tothe strand having SEQ ID NO:1, which can easily be determined by thoseskilled in the art. Likewise, a nucleic acid sequence complement of anynucleic acid sequence of the present invention refers to the nucleicacid sequence of the nucleic acid strand that is complementary to (i.e.,can form a complete double helix with) the strand for which the sequenceis cited.

It should be noted that since nucleic acid sequencing technology is notentirely error-free, SEQ ID NO:1 and SEQ ID NO:3 (as well as othernucleic acid and protein sequences presented herein) represent apparentnucleic acid sequences of certain nucleic acid molecules encoding equineFc_(ε)Rα proteins of the present invention.

In another embodiment, an equine Fc_(ε)Rα gene can be an allelic variantthat includes a similar but not identical sequence to SEQ ID NO:1, SEQID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8 and/or SEQID NO:11. An allelic variant of an equine Fc_(ε)Rα gene is a gene thatoccurs at essentially the same locus (or loci) in the genome as the geneincluding SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:8 and/or SEQ ID NO:11, but which, due to naturalvariations caused by, for example, mutation or recombination, has asimilar but not identical sequence. Allelic variants typically encodeproteins having similar activity to that of the protein encoded by thegene to which they are being compared. Allelic variants can alsocomprise alterations in the 5′ or 3′ untranslated regions of the gene(e.g., in regulatory control regions). Allelic variants are well knownto those skilled in the art and would be expected to be found within agiven horse since the genome is diploid and/or among a group of two ormore horses. The present invention also includes variants due tolaboratory manipulation, such as, but not limited to, variants producedduring polymerase chain reaction amplification.

The minimal size of a Fc_(ε)Rα protein homolog of the present inventionis a size sufficient to be encoded by a nucleic acid molecule capable offorming a stable hybrid (i.e., hybridize under stringent hybridizationconditions) with the complementary sequence of a nucleic acid moleculeencoding the corresponding natural protein. As such, the size of thenucleic acid molecule encoding such a protein homolog is dependent onnucleic acid composition and percent homology between the nucleic acidmolecule and complementary sequence. It should also be noted that theextent of homology required to form a stable hybrid can vary dependingon whether the homologous sequences are interspersed throughout thenucleic acid molecules or are clustered (i.e., localized) in distinctregions on the nucleic acid molecules. The minimal size of such nucleicacid molecules is typically at least about 12 to about 15 nucleotides inlength if the nucleic acid molecules are GC-rich and at least about 15to about 17 bases in length if they are AT-rich. As such, the minimalsize of a nucleic acid molecule used to encode an equine Fc_(ε)Rαprotein homolog of the present invention is from about 12 to about 18nucleotides in length. Thus, the minimal size of an equine Fc_(ε)Rαprotein homolog of the present invention is from about 4 to about 6amino acids in length. There is no limit, other than a practical limit,on the maximal size of such a nucleic acid molecule in that the nucleicacid molecule can include a portion of a gene, an entire gene, multiplegenes, or portions thereof. The preferred size of a protein encoded by anucleic acid molecule of the present invention depends on whether afull-length, fusion, multivalent, or functional portion of such aprotein is desired. Preferably, the preferred size of a protein encodedby a nucleic acid molecule of the present invention is a portion of theprotein that binds to IgE which is about 30 amino acids, more preferablyabout 35 amino acids and even more preferably about 44 amino acids inlength.

As used herein, an equine refers to any member of the horse family.Examples of horses from which to isolate equine Fc_(ε)Rα proteins of thepresent invention (including isolation of the natural protein orproduction of the protein by recombinant or synthetic techniques)include, but are not limited to domestic horses and wild horses, withdomestic horses, including race horses being more preferred.

Suitable horse cells from which to isolate an equine Fc_(ε)Rα protein ofthe present invention include cells that have Fc_(ε)Rα proteins.Preferred horse cells from which to obtain an equine Fc_(ε)Rα protein ofthe present invention include basophil cells, mast cells, mastocytomacells, dendritic cells, B lymphocytes, macrophages, eosinophils, and/ormonocytes. An equine Fc_(ε)Rα of the present invention is preferablyobtained from mastocytoma cells, mast cells or basophil cells.

The present invention also includes mimetopes of equine Fc_(ε)Rαproteins of the present invention. As used herein, a mimetope of anequine Fc_(ε)Rα protein of the present invention refers to any compoundthat is able to mimic the activity of such an equine Fc_(ε)Rα protein(e.g., ability to bind to IgE), often because the mimetope has astructure that mimics the equine Fc_(ε)Rα protein. It is to be noted,however, that the mimetope need not have a structure similar to anequine Fc_(ε)Rα protein as long as the mimetope functionally mimics theprotein. Mimetopes can be, but are not limited to: peptides that havebeen modified to decrease their susceptibility to degradation;anti-idiotypic and/or catalytic antibodies, or fragments thereof;non-proteinaceous immunogenic portions of an isolated protein (e.g.,carbohydrate structures); synthetic or natural organic or inorganicmolecules, including nucleic acids; and/or any other peptidomimeticcompounds. Mimetopes of the present invention can be designed usingcomputer-generated structures of equine Fc_(ε)Rα proteins of the presentinvention. Mimetopes can also be obtained by generating random samplesof molecules, such as oligonucleotides, peptides or other organicmolecules, and screening such samples by affinity chromatographytechniques using the corresponding binding partner, (e.g., an equine IgEFc domain or anti-equine Fc_(ε)Rα antibody). A mimetope can also beobtained by, for example, rational drug design. In a rational drugdesign procedure, the three-dimensional structure of a compound of thepresent invention can be analyzed by, for example, nuclear magneticresonance (NMR) or x-ray crystallography. The three-dimensionalstructure can then be used to predict structures of potential mimetopesby, for example, computer modeling. The predicted mimetope structurescan then be produced by, for example, chemical synthesis, recombinantDNA technology, or by isolating a mimetope from a natural source.Specific examples of equine Fc_(ε)Rα mimetopes include anti-idiotypicantibodies, oligonucleotides produced using Selex™ technology, peptidesidentified by random screening of peptide libraries and proteinsidentified by phage display technology. A preferred mimetope is apeptidomimetic compound that is structurally and/or functionally similarto an equine Fc_(ε)Rα protein of the present invention, particularly tothe IgE Fc domain binding site of the equine Fc_(ε)Rα protein. As usedherein, the Fc domain of an antibody refers to the portion of animmunoglobulin that has Fc receptor binding effector function.Typically, the Fc domain of an IgE comprises the CH2 and CH3 domains ofthe heavy chain constant region.

According to the present invention, an equine Fc, Rα molecule of thepresent invention refers to: an equine Fc_(ε)Rα protein, in particular asoluble equine Fc_(ε)Rα protein; an equine Fc_(ε)Rα homolog; an equineFc_(ε)Rα mimetope; an equine Fc_(ε)Rα substrate analog; or an equineFc_(ε)Rα peptide. Preferably, an equine Fc_(ε)Rα molecule binds to IgE.

One embodiment of an equine Fc_(ε)Rα protein of the present invention isa fusion protein that includes an equine Fc_(ε)Rα protein-containingdomain attached to one or more fusion segments. Suitable fusion segmentsfor use with the present invention include, but are not limited to,segments that can: enhance a protein's stability; act as animmunopotentiator to enhance an immune response against an equineFc_(ε)Rα protein; and/or assist purification of an equine Fc_(ε)Rαprotein (e.g., by affinity chromatography). A suitable fusion segmentcan be a domain of any size that has the desired function (e.g., impartsincreased stability, imparts increased immunogenicity to a protein,and/or simplifies purification of a protein). Fusion segments can bejoined to amino and/or carboxyl termini of the equineFc_(ε)Rα-containing domain of the protein and can be susceptible tocleavage in order to enable straight-forward recovery of an equineFc_(ε)Rα protein. Fusion proteins are preferably produced by culturing arecombinant cell transformed with a fusion nucleic acid molecule thatencodes a protein including the fusion segment attached to either thecarboxyl and/or amino terminal end of an equine Fc_(ε)Rα-containingdomain. Preferred fusion segments include a metal binding domain (e.g.,a poly-histidine segment); an immunoglobulin binding domain (e.g.,Protein A; Protein G; T cell; B cell; Fc receptor or complement proteinantibody-binding domains); a sugar binding domain (e.g., a maltosebinding domain); a “tag” domain (e.g., at least a portion ofβ-galactosidase, a strep tag peptide, other domains that can be purifiedusing compounds that bind to the domain, such as monoclonal antibodies);and/or a linker and enzyme domain (e.g., alkaline phosphatase domainconnected to an equine Fc_(ε)Rα protein by a linker). More preferredfusion segments include metal binding domains, such as a poly-histidinesegment; a maltose binding domain; a strep tag peptide, such as thatavailable from Biometra in Tampa, Fla.; and a phage T7 S10 peptide.

A preferred equine Fc_(ε)Rα protein of the present invention is encodedby a nucleic acid molecule that hybridizes under stringent hybridizationconditions with at least one of the following nucleic acid molecules:neqFc_(ε)Rα₁₀₁₅, neqFc_(ε)Rα₇₆₅, neqFc_(ε)Rα₇₀₈ and neqFc_(ε)Rα₆₀₃.Preferably, the equine Fc_(ε)Rα protein binds to IgE. A furtherpreferred isolated protein is encoded by a nucleic acid molecule thathybridizes under stringent hybridization conditions with a nucleic acidmolecule having nucleic acid sequence SEQ ID NO:3, SEQ ID NO:5 and SEQID NO:8.

Translation of SEQ ID NO:1 suggests that nucleic acid moleculeneqFc_(ε)Rα₁₀₁₅ encodes a full-length equine protein of about 255 aminoacids, referred to herein as PequFc_(ε)Rα₂₅₅, represented by SEQ IDNO:2, assuming an open reading frame having an initiation (start) codonspanning from nucleotide 12 through nucleotide 14 of SEQ ID NO:1 and atermination (stop) codon spanning from nucleotide 777 through nucleotide779 of SEQ ID NO:1. The coding region encoding PequFc_(ε)Rα₂₆₃ isrepresented by nucleic acid molecule neqFc_(ε)Rα₇₆₅, having a codingstrand with the nucleic acid sequence represented by SEQ ID NO:4 and acomplementary strand with the nucleic acid sequence represented by SEQID NO:5. Analysis of SEQ ID NO:2 suggests the presence of a signalpeptide encoded by a stretch of amino acids spanning from amino acid 1through amino acid 19. The proposed mature protein, denoted herein asPequFc_(ε)Rα₂₃₆, contains about 236 amino acids which is representedherein as SEQ ID NO:7. PequFc_(ε)Rα₂₃₆ is encoded by nucleic acidmolecule neqFc_(ε)Rα₇₀₈, having a coding strand with the nucleic acidsequence represented by SEQ ID NO:6 and a complementary strand with thenucleic acid sequence represented by SEQ ID NO:8. The amino acidsequence of PequFc_(ε)Rα₂₃₆ (i.e. SEQ ID NO:7) predicts thatPequFc_(ε)Rα₂₃₆ has an estimated molecular weight of about 27.3 kD, anestimated pI of about 9.77.

Comparison of amino acid sequence SEQ ID NO:2 (i.e., the amino acidsequence of PequFc_(ε)Rα₂₅₅) with amino acid sequences reported inGenBank™ indicates that SEQ ID NO:2 showed the most homology, i.e.,about 61% identity, with a human high affinity IgE receptor α-subunit(SwissProt accession number P12319).

More preferred equine Fc_(ε)Rα proteins of the present invention includeproteins comprising amino acid sequences that are at least about 65%,preferably at least about 70%, more preferably at least about 75%, morepreferably at least about 80%, more preferably at least about 85%, morepreferably at least about 90% and even more preferably about 95%,identical to amino acid sequence SEQ ID NO:2, SEQ ID NO:7 and/or SEQ IDNO:12. Amino acid sequence analysis can be performed using either theDNAsis™ program (available from Hitachi Software, San Bruno, Calif.) orthe MacVector™ program (available from the Eastman Kodak Company, NewHaven, Conn.), preferably using default stringency parameters.

More preferred equine Fc_(ε)Rα proteins of the present invention includeproteins encoded by a nucleic acid molecule comprising at least aportion of neqFc_(ε)Rα₁₀₁₅, neqFc_(ε)Rα₇₆₅, neqFc_(ε)Rα₇₀₈ and/orneqFc_(ε)Rα₆₀₃, or of allelic variants of such nucleic acid molecules,the portion being capable of binding to IgE. More preferred is an equineFc_(ε)Rα protein encoded by neqFc_(ε)Rα₁₀₁₅, neqFc_(ε)Rα₇₆₅,neqFc_(ε)Rα₇₀₈ and/or neqFc_(ε)Rα₆₀₃, or by an allelic variant of suchnucleic acid molecules. Particularly preferred equine Fc_(ε)Rα proteinsare PequFc_(ε)Rα₂₅₅, PequFc_(ε)Rα₂₃₆ and PequFc_(ε)Rα₂₀₁.

In one embodiment, a preferred equine Fc_(ε)Rα protein of the presentinvention is encoded by at least a portion of SEQ ID NO: 1, SEQ ID NO:4,SEQ ID NO:6 and/or SEQ ID NO:11, and, as such, has an amino acidsequence that includes at least a portion of SEQ ID NO:2, SEQ ID NO:7and/or SEQ ID NO:12.

Also preferred is an equine Fc_(ε)Rα protein encoded by an allelicvariant of a nucleic acid molecule comprising at least a portion of SEQID NO:1, SEQ ID NO:4, SEQ ID NO:6 and/or SEQ ID NO:11. Particularlypreferred equine Fc_(ε)Rα proteins of the present invention include SEQIT NO:2, SEQ ID NO:7 and SEQ ID NO:12 (including, but not limited to,the proteins consisting of such sequences, fusion proteins andmultivalent proteins) and proteins encoded by allelic variants ofnucleic acid molecules that encode SEQ ID NO:2, SEQ ID NO:7 and SEQ IDNO:12.

Another embodiment of the present invention is an isolated nucleic acidmolecule that hybridizes under stringent hybridization conditions withan equine Fc_(ε)Rα gene. The identifying characteristics of such a geneare heretofore described. A nucleic acid molecule of the presentinvention can include an isolated natural equine Fc_(ε)Rα gene or ahomolog thereof, the latter of which is described in more detail below.A nucleic acid molecule of the present invention can include one or moreregulatory regions, full-length or partial coding regions, orcombinations thereof. The minimal size of a nucleic acid molecule of thepresent invention is the minimal size that can form a stable hybrid withan equine Fc_(ε)Rα gene under stringent hybridization conditions.

In accordance with the present invention, an isolated nucleic acidmolecule is a nucleic acid molecule that has been removed from itsnatural milieu (i.e., that has been subject to human manipulation) andcan include DNA, RNA, or derivatives of either DNA or RNA. As such,“isolated” does not reflect the extent to which the nucleic acidmolecule has been purified. An isolated equine Fc_(ε)Rα nucleic acidmolecule of the present invention can be isolated from its naturalsource or can be produced using recombinant DNA technology (e.g.,polymerase chain reaction (PCR) amplification, cloning) or chemicalsynthesis. Isolated equine Fc_(ε)Rα nucleic acid molecules can include,for example, natural allelic variants and nucleic acid moleculesmodified by nucleotide insertions, deletions, substitutions, and/orinversions in a manner such that the modifications do not substantiallyinterfere with the nucleic acid molecule's ability to encode an equineFc_(ε)Rα protein of the present invention or to form stable hybridsunder stringent conditions with natural gene isolates.

An equine Fc_(ε)Rα nucleic acid molecule homolog can be produced using anumber of methods known to those skilled in the art (see, for example,Sambrook et al., ibid.). For example, nucleic acid molecules can bemodified using a variety of techniques including, but not limited to,classic mutagenesis and recombinant DNA techniques (e.g., site-directedmutagenesis, chemical treatment, restriction enzyme cleavage, ligationof nucleic acid fragments and/or PCR amplification), synthesis ofoligonucleotide mixtures and ligation of mixture groups to “build” amixture of nucleic acid molecules and combinations thereof. Nucleic acidmolecule homologs can be selected by hybridization with an equineFc_(ε)Rα gene or by screening for function of a protein encoded by thenucleic acid molecule (e.g., ability of an equine Fc_(ε)Rα protein tobind equine IgE).

An isolated nucleic acid molecule of the present invention can include anucleic acid sequence that encodes at least one equine Fc_(ε)Rα proteinof the present invention, examples of such proteins being disclosedherein. Although the phrase “nucleic acid molecule” primarily refers tothe physical nucleic acid molecule and the phrase “nucleic acidsequence” primarily refers to the sequence of nucleotides on the nucleicacid molecule, the two phrases can be used interchangeably, especiallywith respect to a nucleic acid molecule, or a nucleic acid sequence,being capable of encoding an equine Fc_(ε)Rα protein.

One embodiment of the present invention is an equine Fc_(ε)Rα nucleicacid molecule that hybridizes under stringent hybridization conditionswith nucleic acid molecule neqFc_(ε)Rα₁₀₁₅ and preferably with a nucleicacid molecule having nucleic acid sequence SEQ ID NO:1 and/or SEQ IDNO:3.

Comparison of nucleic acid sequence SEQ ID NO:1 (i.e., the nucleic acidsequence of the coding strand of neqFc_(ε)Rα₁₀₁₅) with nucleic acidsequences reported in GenBank indicates that SEQ ID NO:1 showed the mosthomology, i.e., about 75% identity to a human mRNA for immunoglobulin Ereceptor alpha chain gene (Accession number X06948).

Preferred equine Fc_(ε)Rα nucleic acid molecules include nucleic acidmolecules having a nucleic acid sequence that is at least about 80%,preferably at least about 85%, more preferably at least about 90%, andeven more preferably at least about 95% identical to nucleic acidsequence SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:8 and/or SEQ ID NO:11. DNA sequence analysis can beperformed using either the DNAsis™ program or the MacVector™ program,preferably using default stringency parameters.

Another preferred nucleic acid molecule of the present inventionincludes at least a portion of nucleic acid sequence SEQ ID NO:1, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8 and/or SEQ IDNO:11, that is capable of hybridizing to an equine Fc_(ε)Rα gene of thepresent invention, as well as allelic variants thereof. A more preferrednucleic acid molecule includes the nucleic acid sequence SEQ ID NO:1,SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8 and/orSEQ ID NO:11, as well as allelic variants of such a nucleic acidmolecule. Such nucleic acid molecules can include nucleotides inaddition to those included in the SEQ ID NOs, such as, but not limitedto, a full-length gene, a full-length coding region, a nucleic acidmolecule encoding a fusion protein, or a nucleic acid molecule encodinga multivalent protective compound.

Preferred equine Fc_(ε)Rα nucleic acid molecules also include nucleicacid molecules having a nucleic acid sequence that is at least about80%, preferably at least about 85%, more preferably at least about 90%,and even more preferably at least about 95% identical to nucleic acidmolecules neqFc_(ε)Rα₁₀₁₅, neqFc_(ε)Rα₇₆₅, neqFc_(ε)Rα₇₀₈ and/orneqFc_(ε)Rα₆₀₃. Particularly preferred nucleic acid molecules includeneqFc_(ε)Rα₁₀₁₅, neqFc_(ε)Rα₇₆₅, neqFc_(ε)Rα₇₀₃ and neqFc_(ε)Rα₆₀₃.

The present invention also includes a nucleic acid molecule encoding aprotein having at least a portion of SEQ ID NO:2, SEQ ID NO:7 and SEQ IDNO:12, including nucleic acid molecules that have been modified toaccommodate codon usage properties of the cells in which such nucleicacid molecules are to be expressed.

Knowing the nucleic acid sequences of certain equine Fc<Rα nucleic acidmolecules of the present invention allows one skilled in the art to, forexample, (a) make copies of those nucleic acid molecules, (b) obtainnucleic acid molecules including at least a portion of such nucleic acidmolecules (e.g., nucleic acid molecules including full-length genes,full-length coding regions, regulatory control sequences, truncatedcoding regions), and (c) obtain equine Fc_(ε)Rα nucleic acid moleculesfrom other horses. Such nucleic acid molecules can be obtained in avariety of ways including screening appropriate expression librarieswith antibodies of the present invention; traditional cloning techniquesusing oligonucleotide probes of the present invention to screenappropriate libraries or DNA; and PCR amplification of appropriatelibraries or DNA using oligonucleotide primers of the present invention.Preferred libraries to screen or from which to amplify nucleic acidmolecule include equine basophil cell, mast cell, mastocytoma cell,dendritic cell, B lymphocyte, macrophage, eosinophil, and/or monocytecDNA libraries as well as genomic DNA libraries. Similarly, preferredDNA sources to screen or from which to amplify nucleic acid moleculesinclude equine basophil cells, mast cells, mastocytoma cells, dendriticcells, B lymphocytes, macrophages, eosinophils, and/or monocytes cDNAand genomic DNA. Techniques to clone and amplify genes are disclosed,for example, in Sambrook et al., ibid.

The present invention also includes nucleic acid molecules that areoligonucleotides capable of hybridizing, under stringent hybridizationconditions, with complementary regions of other, preferably longer,nucleic acid molecules of the present invention such as those comprisingequine Fc_(ε)Rα genes or other equine Fc_(ε)Rα nucleic acid molecules.Oligonucleotides of the present invention can be RNA, DNA, orderivatives of either. The minimum size of such oligonucleotides is thesize required for formation of a stable hybrid between anoligonucleotide and a complementary sequence on a nucleic acid moleculeof the present invention. Minimal size characteristics are disclosedherein. The present invention includes oligonucleotides that can be usedas, for example, probes to identify nucleic acid molecules, primers toproduce nucleic acid molecules or therapeutic reagents to inhibit equineFc_(ε)Rα protein production or activity (e.g., as antisense-, triplexformation-, ribozyme- and/or RNA drug-based reagents). The presentinvention also includes the use of such oligonucleotides to protectanimals from disease using one or more of such technologies. Appropriateoligonucleotide-containing therapeutic compositions can be administeredto an animal using techniques known to those skilled in the art.

One embodiment of the present invention includes a recombinant vector,which includes at least one isolated nucleic acid molecule of thepresent invention, inserted into any vector capable of delivering thenucleic acid molecule into a host cell. Such a vector containsheterologous nucleic acid sequences, that is nucleic acid sequences thatare not naturally found adjacent to nucleic acid molecules of thepresent invention and that preferably are derived from a species otherthan the species from which the nucleic acid molecule(s) are derived.The vector can be either RNA or DNA, either prokaryotic or eukaryotic,and typically is a virus or a plasmid. Recombinant vectors can be usedin the cloning, sequencing, and/or otherwise manipulation of equineFc_(ε)Rα nucleic acid molecules of the present invention.

One type of recombinant vector, referred to herein as a recombinantmolecule, comprises a nucleic acid molecule of the present inventionoperatively linked to an expression vector. The phrase operativelylinked refers to insertion of a nucleic acid molecule into an expressionvector in a manner such that the molecule is able to be expressed whentransformed into a host cell. As used herein, an expression vector is aDNA or RNA vector that is capable of transforming a host cell and ofeffecting expression of a specified nucleic acid molecule. Preferably,the expression vector is also capable of replicating within the hostcell. Expression vectors can be either prokaryotic or eukaryotic, andare typically viruses or plasmids. Expression vectors of the presentinvention include any vectors that function (i.e., direct geneexpression) in recombinant cells of the present invention, including inbacterial, fungal, endoparasite, insect, other animal, and plant cells.Preferred expression vectors of the present invention can direct geneexpression in bacterial, yeast, insect and mammalian cells and morepreferably in the cell types disclosed herein.

In particular, expression vectors of the present invention containregulatory sequences such as transcription control sequences,translation control sequences, origins of replication, and otherregulatory sequences that are compatible with the recombinant cell andthat control the expression of nucleic acid molecules of the presentinvention. In particular, recombinant molecules of the present inventioninclude transcription control sequences. Transcription control sequencesare sequences which control the initiation, elongation, and terminationof transcription. Particularly important transcription control sequencesare those which control transcription initiation, such as promoter,enhancer, operator and repressor sequences. Suitable transcriptioncontrol sequences include any transcription control sequence that canfunction in at least one of the recombinant cells of the presentinvention. A variety of such transcription control sequences are knownto those skilled in the art. Preferred transcription control sequencesinclude those which function in bacterial, yeast, insect and mammaliancells, such as, but not limited to, tac, lac, trp, trc, oxy-pro,omp/lpp, rrnB, bacteriophage lambda (such as lambda p_(L) and lambdap_(R) and fusions that include such promoters), bacteriophage T7, T7lac,bacteriophage T3, bacteriophage SP6, bacteriophage SP01,metallothionein, alpha-mating factor, Pichia alcohol oxidase, alphavirussubgenomic promoters (such as Sindbis virus subgenomic promoters),antibiotic resistance gene, baculovirus, Heliothis zea insect virus,vaccinia virus, herpesvirus, raccoon poxvirus, other poxvirus,adenovirus, cytomegalovirus (such as intermediate early promoters),simian virus 40, retrovirus, actin, retroviral long terminal repeat,Rous sarcoma virus, heat shock, phosphate and nitrate transcriptioncontrol sequences as well as other sequences capable of controlling geneexpression in prokaryotic or eukaryotic cells. Additional suitabletranscription control sequences include tissue-specific promoters andenhancers as well as lymphokine-inducible promoters (e.g., promotersinducible by interferons or interleukins). Transcription controlsequences of the present invention can also include naturally occurringtranscription control sequences naturally associated with horses.

Suitable and preferred nucleic acid molecules to include in recombinantvectors of the present invention are as disclosed herein. Preferrednucleic acid molecules to include in recombinant vectors, andparticularly in recombinant molecules, include neqFc_(ε)Rα₁₀₁₅,neqFc_(ε)Rα₇₆₅, neqFc_(ε)Rα₇₀₈ and neqFc_(ε)Rα₆₀₃. A particularlypreferred recombinant molecule of the present invention includespFB-neqFc_(ε)Rα₆₀₃, the production of which is described in the Examplessection.

Recombinant molecules of the present invention may also (a) containsecretory signals (i.e., signal segment nucleic acid sequences) toenable an expressed equine Fc_(ε)Rα protein of the present invention tobe secreted from the cell that produces the protein and/or (b) containfusion sequences which lead to the expression of nucleic acid moleculesof the present invention as fusion proteins. Examples of suitable signalsegments include any signal segment capable of directing the secretionof a protein of the present invention. Preferred signal segmentsinclude, but are not limited to, tissue plasminogen activator (t-PA)₇interferon, interleukin, growth hormone, histocompatibility and viralenvelope glycoprotein signal segments, as well as natural signalsegments. Suitable fusion segments encoded by fusion segment nucleicacids are disclosed herein. In addition, a nucleic acid molecule of thepresent invention can be joined to a fusion segment that directs theencoded protein to the proteosome, such as a ubiquitin fusion segment.Recombinant molecules may also include intervening and/or untranslatedsequences surrounding and/or within the nucleic acid sequences ofnucleic acid molecules of the present invention.

Another embodiment of the present invention includes a recombinant cellcomprising a host cell transformed with one or more recombinantmolecules of the present invention. Transformation of a nucleic acidmolecule into a cell can be accomplished by any method by which anucleic acid molecule can be inserted into the cell. Transformationtechniques include, but are not limited to, transfection,electroporation, microinjection, lipofection, adsorption, and protoplastfusion. A recombinant cell may remain unicellular or may grow into atissue, organ or a multicellular organism. Transformed nucleic acidmolecules of the present invention can remain extrachromosomal or canintegrate into one or more sites within a chromosome of the transformed(i.e., recombinant) cell in such a manner that their ability to beexpressed is retained. Preferred nucleic acid molecules with which totransform a cell include equine Fc_(ε)Rα nucleic acid moleculesdisclosed herein. Particularly preferred nucleic acid molecules withwhich to transform a cell include neqFc_(ε)Rα₁₀₁₅, neqFc_(ε)Rα₇₆₅,neqFc_(ε)Rα₇₀₈ and neqFc_(ε)Rα₆₀₃.

Suitable host cells to transform include any cell that can betransformed with a nucleic acid molecule of the present invention. Hostcells can be either untransformed cells or cells that are alreadytransformed with at least one nucleic acid molecule (e.g., nucleic acidmolecules encoding one or more proteins of the present invention and/orother proteins useful in the production of multivalent vaccines). Hostcells of the present invention either can be endogenously (i.e.,naturally) capable of producing equine PFc_(ε)Rα proteins of the presentinvention or can be capable of producing such proteins after beingtransformed with at least one nucleic acid molecule of the presentinvention. Host cells of the present invention can be any cell capableof producing at least one protein of the present invention, and includebacterial, fungal (including yeast), other insect, other animal andplant cells. Preferred host cells include bacterial, mycobacteria,yeast, parasite, insect and mammalian cells. More preferred host cellsinclude Salmonella, Escherichia, Bacillus, Listeria, Saccharomyces,Spodoptera, Mycobacteria, Trichoplusia, BHK (baby hamster kidney) cells,MDCK cells (normal dog kidney cell line for canine herpesviruscultivation), CRFK cells (normal cat kidney cell line for felineherpesvirus cultivation), CV-1 cells (African monkey kidney cell lineused, for example, to culture raccoon poxvirus), COS (e.g., COS-7)cells, and Vero cells. Particularly preferred host cells are Escherichiacoli, including E. coli K-12 derivatives; Salmonella typhi; Salmonellatyphimurium, including attenuated strains such as UK-1_(X)3987 andSR-11_(X)4072; Spodoptera frugiperda; Trichoplusia ni; BHK cells; MDCKcells; CRFK cells; CV-1 cells; COS cells; Vero cells; andnon-tumorigenic mouse myoblast G8 cells (e.g., ATCC CRL 1246).Additional appropriate mammalian cell hosts include other kidney celllines, other fibroblast cell lines (e.g., human, murine or chickenembryo fibroblast cell lines), myeloma cell lines, Chinese hamster ovarycells, mouse NIH/3T3 cells, LMTK³¹ cells and/or HeLa cells. In oneembodiment, the proteins may be expressed as heterologous proteins inmyeloma cell lines employing immunoglobulin promoters.

A recombinant cell is preferably produced by transforming a host cellwith one or more recombinant molecules, each comprising one or morenucleic acid molecules of the present invention operatively linked to anexpression vector containing one or more transcription controlsequences. The phrase operatively linked refers to insertion of anucleic acid molecule into an expression vector in a manner such thatthe molecule is able to be expressed when transformed into a host cell.

A recombinant molecule of the present invention is a molecule that caninclude at least one of any nucleic acid molecule heretofore describedoperatively linked to at least one of any transcription control sequencecapable of effectively regulating expression of the nucleic acidmolecule(s) in the cell to be transformed, examples of which aredisclosed herein. A particularly preferred recombinant molecule includespFB-neqFc_(ε)Rα₆₀₃.

A recombinant cell of the present invention includes any celltransformed with at least one of any nucleic acid molecule of thepresent invention. Suitable and preferred nucleic acid molecules as wellas suitable and preferred recombinant molecules with which to transformcells are disclosed herein. A particularly preferred recombinant cellincludes S. frugiperda:pFB-neqFc_(ε)Rα₆₀₃. Details regarding theproduction of this recombinant cell is disclosed herein.

Recombinant DNA technologies can be used to improve expression oftransformed nucleic acid molecules by manipulating, for example, thenumber of copies of the nucleic acid molecules within a host cell, theefficiency with which those nucleic acid molecules are transcribed, theefficiency with which the resultant transcripts are translated, and theefficiency of post-translational modifications. Recombinant techniquesuseful for increasing the expression of nucleic acid molecules of thepresent invention include, but are not limited to, operatively linkingnucleic acid molecules to high-copy number plasmids, integration of thenucleic acid molecules into one or more host cell chromosomes, additionof vector stability sequences to plasmids, substitutions ormodifications of transcription control signals (e.g., promoters,operators, enhancers), substitutions or modifications of translationalcontrol signals (e.g., ribosome binding sites, Shine-Dalgarnosequences), modification of nucleic acid molecules of the presentinvention to correspond to the codon usage of the host cell, deletion ofsequences that destabilize transcripts, and use of control signals thattemporally separate recombinant cell growth from recombinant enzymeproduction during fermentation. The activity of an expressed recombinantprotein of the present invention may be improved by fragmenting,modifying, or derivatizing nucleic acid molecules encoding such aprotein.

Isolated equine. Fc_(ε)Rα proteins of the present invention can beproduced in a variety of ways, including production and recovery ofnatural proteins, production and recovery of recombinant proteins, andchemical synthesis of the proteins. In one embodiment, an isolatedprotein of the present invention is produced by culturing a cell capableof expressing the protein under conditions effective to produce theprotein, and recovering the protein. A preferred cell to culture is arecombinant cell of the present invention. Effective culture conditionsinclude, but are not limited to, effective media, bioreactor,temperature, pH and oxygen conditions that permit protein production. Aneffective medium refers to any medium in which a cell is cultured toproduce an equine Fc_(ε)Rα protein of the present invention. Such amedium typically comprises an aqueous medium having assimilable carbon,nitrogen and phosphate sources, and appropriate salts, minerals, metalsand other nutrients, such as vitamins. Cells of the present inventioncan be cultured in conventional fermentation bioreactors, shake flasks,test tubes, microtiter dishes, and petri plates. Culturing can becarried out at a temperature, pH and oxygen content appropriate for arecombinant cell. Such culturing conditions are within the expertise ofone of ordinary skill in the art. Examples of suitable conditions areincluded in the Examples section.

Depending on the vector and host system used for production, resultantproteins of the present invention may either remain within therecombinant cell; be secreted into the fermentation medium; be secretedinto a space between two cellular membranes, such as the periplasmicspace in E. coli; or be retained on the outer surface of a cell or viralmembrane. The phrase “recovering the protein”, as well as similarphrases, refers to collecting the whole fermentation medium containingthe protein and need not imply additional steps of separation orpurification. Proteins of the present invention can be purified using avariety of standard protein purification techniques, such as, but notlimited to, affinity chromatography, ion exchange chromatography,filtration, electrophoresis, hydrophobic interaction chromatography, gelfiltration chromatography, reverse phase chromatography, concanavalin Achromatography, chromatofocusing and differential solubilization.Proteins of the present invention are preferably retrieved in“substantially pure” form. As used herein, “substantially pure” refersto a purity that allows for the effective use of the protein as atherapeutic composition or diagnostic. A therapeutic composition foranimals, for example, should exhibit no substantial.

The present invention also includes isolated (i.e., removed from theirnatural milieu) antibodies that selectively bind to an equine Fc_(ε)Rαprotein of the present invention or a mimetope thereof (i.e.,anti-equine Fc_(ε)Rα antibodies). As used herein, the term “selectivelybinds to” an equine Fc_(ε)Rα protein refers to the ability of antibodiesof the present invention to preferentially bind to specified proteinsand mimetopes thereof of the present invention. Binding can be measuredusing a variety of methods standard in the art including enzymeimmunoassays (e.g., ELISA), immunoblot assays, etc.; see, for example,Sambrook et al., ibid. An anti-equine Fc_(ε)Rα antibody preferablyselectively binds to an equine Fc_(ε)Rα protein in such a way as toreduce the activity of that protein.

Isolated antibodies of the present invention can include antibodies in abodily fluid (such as, but not limited to, serum), or antibodies thathave been purified to varying degrees. Antibodies of the presentinvention can be polyclonal or monoclonal. Functional equivalents ofsuch antibodies, such as antibody fragments and genetically-engineeredantibodies (including single chain antibodies or chimeric antibodiesthat can bind to more than one epitope) are also included in the presentinvention.

A preferred method to produce antibodies of the present inventionincludes (a) administering to an animal an effective amount of aprotein, peptide or mimetope thereof of the present invention to producethe antibodies and (b) recovering the antibodies. In another method,antibodies of the present invention are produced recombinantly usingtechniques as heretofore disclosed to produce equine Fc_(ε)Rα proteinsof the present invention. Antibodies raised against defined proteins ormimetopes can be advantageous because such antibodies are notsubstantially contaminated with antibodies against other substances thatmight otherwise cause interference in a diagnostic assay or side effectsif used in a therapeutic composition.

Antibodies of the present invention have a variety of potential usesthat are within the scope of the present invention. For example, suchantibodies can be used (a) as tools to detect Fc epsilon receptor in thepresence or absence of IgE and/or (b) as tools to screen expressionlibraries and/or to recover desired proteins of the present inventionfrom a mixture of proteins and other contaminants. Furthermore,antibodies of the present invention can be used to target cytotoxicagents to cells having Fc epsilon receptors such as those disclosedherein in order to directly kill such cells. Targeting can beaccomplished by conjugating (i.e., stably joining) such antibodies tothe cytotoxic agents using techniques known to those skilled in the art.Suitable cytotoxic agents are known to those skilled in the art.Antibodies of the present invention, including Fc_(ε)Rα-binding portionsthereof, can also be used, for example, to inhibit binding of IgE to Fcepsilon receptors, to produce anti-equine Fc_(ε)Rα idiotypic antibodies,to purify cells having equine Fc_(ε)Rα proteins, to stimulateintracellular signal transduction through an equine Fc_(ε)Rα and toidentify cells having equine Fc_(ε)Rα proteins.

An equine Fc_(ε)Rα molecule of the present invention can includechimeric molecules comprising a portion of an equine Fc_(ε)Rα moleculethat binds to an IgE and a second molecule that enables the chimericmolecule to be bound to a substrate in such a manner that the Fc_(ε)Rαmolecule portion binds to IgE in essentially the same manner as aFc_(ε)Rα molecule that is not bound to a substrate. An example of asuitable second molecule includes a portion of an immunoglobulinmolecule or another ligand that has a suitable binding partner that canbe immobilized on a substrate, e.g., biotin and avidin, or ametal-binding protein and a metal (e.g., H is), or a sugar-bindingprotein and a sugar (e.g., maltose).

An equine Fc_(ε)Rα molecule of the present invention can includechimeric molecules comprising a portion of an equine Fc_(ε)Rα moleculethat binds to an IgE and a second molecule, such as an enzyme, thatenables the chimeric molecule to bind to IgE in essentially the samemanner as a Fc_(ε)Rα molecule which does not include such a secondmolecule, and to hydrolyze a substrate in such a manner so as to give adetectable signal. An example of a suitable second molecule includesalkaline phosphatase, horse radish peroxidase or urease. In oneembodiment an equine Fc_(ε)Rα chimeric molecule of the present inventioncomprises a protein encoded by a recombinant molecule comprising anucleic acid molecule that encodes at least a portion of an equineFc_(ε)Rα molecule that binds to an IgE, operatively linked to a nucleicacid molecule that encodes an enzyme, preferably alkaline phosphatase.

An equine Fc_(ε)Rα molecule of the present invention can be contained ina formulation, herein referred to as a Fc_(ε)Rα molecule formulation.For example, an equine Fc_(ε)Rα molecule can be combined with a bufferin which the equine Fc_(ε)Rα molecule is solubilized, and/or with acarrier. Suitable buffers and carriers are known to those skilled in theart. Examples of suitable buffers include any buffer in which an equineFc_(ε)Rα molecule can function to selectively bind to IgE, such as, butnot limited to, phosphate buffered saline, water, saline, phosphatebuffer, bicarbonate buffer, HEPES buffer(N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid buffered saline),TES buffer (Tris-EDTA buffered saline), Tris buffer and TAE buffer(Tris-acetate-EDTA). Examples of carriers include, but are not limitedto, polymeric matrices, toxoids, and serum albumins, such as bovineserum albumin. Carriers can be mixed with equine Fc_(ε)Rα molecules orconjugated (i.e., attached) to equine Fc_(ε)Rα molecules in such amanner as to not substantially interfere with the ability of the equineFc_(ε)Rα molecules to selectively bind to IgE.

An equine Fc_(ε)Rα protein of the present invention can be bound to thesurface of a cell comprising the equine Fc_(ε)Rα protein. A preferredequine Fc_(ε)Rα protein-bearing cell includes a recombinant cellcomprising a nucleic acid molecule encoding an equine Fc_(ε)Rα proteinof the present invention. A more preferred recombinant cell of thepresent invention comprises a nucleic acid molecule that encodes atleast one of the following proteins: PequFc_(ε)Rα₂₅₅, PequFc_(ε)Rα₂₃₆and PequFc_(ε)Rα₂₀₁. An even more preferred recombinant cell comprises anucleic acid molecule including neqFc_(ε)Rα₁₀₁₅, neqFc_(ε)Rα₇₆₅,neqFc_(ε)Rα₇₀₈ and neqFc_(ε)Rα₆₀₃ with a recombinant cell comprising anucleic acid molecule comprising a nucleic acid sequence including SEQID NO:1, SEQ ID NO:4, SEQ ID NO:6 or SEQ ID NO:11, or a nucleic acidmolecule comprising an allelic variant of a nucleic acid moleculecomprising SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:6 or SEQ ID NO:11, beingeven more preferred.

In addition, an equine Fc_(ε)Rα molecule formulation of the presentinvention can include not only an equine Fc_(ε)Rα molecule but also oneor more additional antigens or antibodies useful in detecting IgE. Asused herein, an antigen refers to any molecule capable of beingselectively bound by an antibody. As used herein, selective binding of afirst molecule to a second molecule refers to the ability of the firstmolecule to preferentially bind (e.g., having higher affinity higheravidity) to the second molecule when compared to the ability of a firstmolecule to bind to a third molecule. The first molecule need notnecessarily be the natural ligand of the second molecule. Examples ofsuch antibodies include, but are not limited to, antibodies that bindselectively to the constant region of an IgE heavy (i.e., anti-IgEisotype antibody) or antibodies that bind selectively to an IgE having aspecific antigen specificity (i.e., anti-IgE idiotypic antibody).Suitable anti-IgE antibodies for use in a formulation of the presentinvention are not capable of cross-linking two or more IgE antibodies.Preferred anti-IgE antibodies include Fab fragments of the antibodies(as defined in Janeway et al., ibid.). Examples of such antigens includeany antigen known to induce the production of IgE. Preferred antigensinclude allergens and parasite antigens. Allergens include, but are notlimited to allergens ingested, inhaled or contacted by a horse.Allergens of the present invention are preferably derived from fungi,rusts, smuts, bacteria, trees, weeds, shrubs, grasses, wheat, corn,grains, hays, straws, oats, alfalfa, clovers, soybeans, yeasts, fleas,flies, mosquitoes, mites, midges, biting gnats, lice, bees, wasps, ants,true bugs or ticks. A suitable biting gnat allergen includes an allergenderived from a gnat, in particular a gnat saliva antigen. A preferredgnat allergen includes a gnat saliva antigen, in particular a gnatsaliva antigen derived from a gnat of the genus Culicoides. A suitableflea allergen includes an allergen derived from a flea, in particularflea saliva antigen. A preferred flea allergen includes a flea salivaantigen. Preferred flea saliva antigens include antigens such as thosedisclosed in PCT Patent Publication No. WO 96/11271, published Apr. 18,1996, by Frank et al. (this publication is incorporated by referenceherein in its entirety), with flea saliva products and flea salivaproteins being particularly preferred. According to the presentinvention, a flea saliva protein includes a protein produced byrecombinant DNA methods, as well as proteins isolated by other methodsdisclosed in PCT Patent Publication No. WO 96/11271.

Preferred general allergens include those derived from grass, MeadowFescue, curly dock, plantain, Mexican firebush, lamb's quarters,pigweed, ragweed, goldenrod, sorrel, legumes, dandelion, sage, elm,cocklebur, elder, walnut, maple, sycamore, hickory, aspen, pine,cottonwood, ash, birch, cedar, oak, mulberry, cockroach,Dermataphagoides, Alternaria, Aspergillus, Cladosporium, Fusarium,Helminthosporium, Mucor, Curvularia, Candida, Penicillium, Pullularia,Rhizopus and/or Tricophyton. More preferred general allergens includethose derived from Johnson grass, Kentucky blue grass, meadow fescue,orchard grass, perennial rye grass, red top grass, timothy grass,Bermuda grass, salt grass, brome grass, curly dock, yellow dock, Englishplantain, Mexican firebush, lamb's quarters, rough pigweed, shortragweed, goldenrod, sheep sorrel, red clover, dandelion, wormwood sage,American elm, common cocklebur, box elder, marsh elder, black walnut,red maple, eastern sycamore, white pine, eastern cottonwood, green ash,river birch, red cedar, red oak, red mulberry, cockroach, grain smut,oat stem rust, wheat stem rust, Dermataphagoides farinae, Alternariaalternate, Alternaria tenuis, Curvularia spicifera, Aspergillusfumigatus, Cladosporium herbarum, Fusarium vasinfectum, Helminthosporiumsativum, Mucor recemosus, Penicillium notatum, Pullularia pullulans,Rhizopus nigricans and/or Tricophyton spp. The term “derived from”refers to a natural allergen of such plants or organisms (i.e., anallergen directly isolated from such plants or organisms), as well as,non-natural allergens of such plants or organisms that posses at leastone epitope capable of eliciting an immune response against an allergen(e.g., produced using recombinant DNA technology or by chemicalsynthesis). Preferred allergens include those that cause allergicrespiratory diseases in equines, including, for example, chronicobstructive pulmonary disease, exercise induced pulmonary hemorrhage andinhalant-induced urticaria. Such allergens include, but are not limitedto, molds, components of dust and components of feed.

One embodiment of the present invention is a method to detect IgE whichincludes the steps of: (a) contacting an isolated equine Fc_(ε)Rαmolecule with a putative IgE-containing composition under conditionssuitable for formation of an equine Fc_(ε)Rα molecule:IgE complex; and(b) detecting the presence of IgE by detecting the equine Fc_(ε)Rαmolecule:IgE complex. Presence of such an equine Fc_(ε)Rα molecule:IgEcomplex indicates that the animal is producing IgE. Preferred IgE todetect using an equine Fc_(ε)Rα molecule include equine IgE, canine IgE,feline IgE and human IgE, with equine IgE being particularly preferred.The present method can further include the step of determining whetheran IgE complexed with an equine Fc_(ε)Rα protein is heat labile.Preferably, a heat labile IgE is determined by incubating an IgE atabout 56° C. for about 3 or about 4 hours. Without being bound bytheory, the inventors believe that heat labile forms of IgE bind tocertain allergens and non-heat labile forms of IgE bind to other typesof allergens. As such, detection of heat labile IgE compared withnon-heat labile IgE can be used to discriminate between allergensensitivities.

As used herein, canine refers to any member of the dog family, includingdomestic dogs, wild dogs and zoo dogs. Examples of dogs include, but arenot limited to, domestic dogs, wild dogs, foxes, wolves, jackals andcoyotes. As used herein, feline refers to any member of the cat family,including domestic cats, wild cats and zoo cats. Examples of catsinclude, but are not limited to, domestic cats, wild cats, lions,tigers, leopards, panthers, cougars, bobcats, lynx, jaguars, cheetahs,and servals.

As used herein, the term “contacting” refers to combining or mixing, inthis case a putative IgE-containing composition with an equine Fc_(ε)Rαmolecule. Formation of a complex between an equine Fc_(ε)Rα molecule andan IgE refers to the ability of the equine Fc_(ε)Rα molecule toselectively bind to the IgE in order to form a stable complex that canbe measured (i.e., detected). As used herein, the term selectively bindsto an IgE refers to the ability of an equine Fc_(ε)Rα molecule of thepresent invention to preferentially bind to IgE, without being able tosubstantially bind to other antibody isotypes. Binding between an equineFc_(ε)Rα molecule and an IgE is effected under conditions suitable toform a complex; such conditions (e.g., appropriate concentrations,buffers, temperatures, reaction times) as well as methods to optimizesuch conditions are known to those skilled in the art, and examples aredisclosed herein. Examples of complex formation conditions are alsodisclosed in, for example, in Sambrook et al., ibid.

As used herein, the term “detecting complex formation” refers todetermining if any complex is formed, i.e., assaying for the presence(i.e., existence) of a complex. If complexes are formed, the amount ofcomplexes formed can, but need not be, determined. Complex formation, orselective binding, between equine Fc_(ε)Rα molecule and any IgE in thecomposition can be measured (i.e., detected, determined) using a varietyof methods standard in the art (see, for example, Sambrook et al.ibid.), examples of which are disclosed herein.

In one embodiment, a putative IgE-containing composition of the presentmethod includes a biological sample from an animal. A suitablebiological sample includes, but is not limited to, a bodily fluidcomposition or a cellular composition. A bodily fluid refers to anyfluid that can be collected (i.e., obtained) from an animal, examples ofwhich include, but are not limited to, blood, serum, plasma, urine,tears, aqueous humor, cerebrospinal fluid (CSF), saliva, lymph, nasalsecretions, tracheobronchial aspirates, milk, feces and fluids obtainedthrough bronchial alveolar lavage. Such a composition of the presentmethod can, but need not be, pretreated to remove at least some of thenon-IgE isotypes of immunoglobulin and/or other proteins, such asalbumin, present in the fluid. Such removal can include, but is notlimited to, contacting the bodily fluid with a material, such as ProteinG, to remove IgG antibodies and/or affinity purifying IgE antibodiesfrom other components of the body fluid by exposing the fluid to, forexample, Concanavalin A. In another embodiment, a composition includescollected bodily fluid that is pretreated to concentrate immunoglobulincontained in the fluid. For example, immunoglobulin contained in abodily fluid can be precipitated from other proteins using ammoniumsulfate. A preferred composition of the present method is serum.

In another embodiment, a IgE-containing composition of the presentmethod includes a cell that produces IgE. Such a cell can have IgE boundto the surface of the cell and/or can secrete IgE. An example of such acell includes myeloma cells. IgE can be bound to the surface of a celleither directly to the membrane of the cell or bound to a molecule(e.g., an antigen) bound to the surface of the cell.

A complex can be detected in a variety of ways including, but notlimited to use of one or more of the following assays: an enzyme-linkedimmunoassay, a radioimmunoassay, a fluorescence immunoassay, achemiluminescent assay, a lateral flow assay, an agglutination assay, aparticulate-based assay (e.g., using particulates such as, but notlimited to, magnetic particles or plastic polymers, such as latex orpolystyrene beads), an immunoprecipitation assay, a BioCore™ assay(e.g., using colloidal gold) and an immunoblotting assay (e.g., awestern blot). Such assays are well known to those skilled in the art.Assays can be used to give qualitative or quantitative results dependingon how they are used. Some assays, such as agglutination, particulateseparation, and immunoprecipitation, can be observed visually (e.g.,either by eye or by a machines, such as a densitometer orspectrophotometer) without the need for a detectable marker. In otherassays, conjugation (i.e., attachment) of a detectable marker to theequine Fc_(ε)Rα molecule or to a reagent that selectively binds to theequine Fc_(ε)Rα molecule or to the IgE being detected (described in moredetail below) aids in detecting complex formation. Examples ofdetectable markers include, but are not limited to, a radioactive label,an enzyme, a fluorescent label, a chemiluminescent label, a chromophoriclabel or a ligand. A ligand refers to a molecule that binds selectivelyto another molecule. Preferred detectable markers include, but are notlimited to, fluorescein, a radioisotope, a phosphatase (e.g., alkalinephosphatase), biotin, avidin, a peroxidase (e.g., horseradishperoxidase) and biotin-related compounds or avidin-related compounds(e.g., streptavidin or ImmunoPure® NeutrAvidin available from Pierce,Rockford, Ill.). According to the present invention, a detectable markercan be connected to an equine Fc_(ε)Rα molecule using, for example,chemical conjugation or recombinant DNA technology (e.g., connection ofa fusion segment such as that described herein for a metal bindingdomain; an immunoglobulin binding; a sugar binding domain; and a “tag”domain). Preferably a carbohydrate group of the equine Fc_(ε)Rα moleculeis chemically conjugated to biotin.

In one embodiment, a complex is detected by contacting a putativeIgE-containing composition with an equine Fc_(ε)Rα molecule that isconjugated to a detectable marker. A suitable detectable marker toconjugate to an equine Fc_(ε)Rα molecule includes, but is not limitedto, a radioactive label, a fluorescent label, an enzyme label, achemiluminescent label, a chromophoric label or a ligand. A detectablemarker is conjugated to an equine Fc_(ε)Rα molecule in such a manner asnot to block the ability of the equine Fc_(ε)Rα molecule to bind to theIgE being detected. Preferably, a carbohydrate group of an equineFc_(ε)Rα molecule is conjugated to biotin.

In another embodiment, an equine Fc_(ε)Rα molecule:IgE complex isdetected by contacting a putative IgE-containing composition with anequine Fc_(ε)RαC molecule and then contacting the complex with anindicator molecule. Suitable indicator molecules of the presentinvention include molecules that can bind to either the equine Fc_(ε)Rαmolecule or to the IgE antibody. As such, an indicator molecule cancomprise, for example, an antigen, an antibody and a lectin, dependingupon which portion of the equine Fc_(ε)Rα molecule:IgE complex is beingdetected. Preferred indicator molecules that are antibodies include, forexample, anti-IgE antibodies and anti-equine Fc_(ε)Rα antibodies.Preferred lectins include those lectins that bind to high-mannosegroups. More preferred lectins bind to high-mannose groups present on anequine Fc_(ε)Rα protein of the present invention produced in insectcells. An indicator molecule itself can be attached to a detectablemarker of the present invention. For example, an antibody can beconjugated to biotin, horseradish peroxidase, alkaline phosphatase orfluorescein.

In one preferred embodiment, an equine Fc_(ε)Rα molecule:IgE complex isdetected by contacting the complex with an indicator molecule thatselectively binds to an equine Fc_(ε)Rα molecule of the presentinvention. Examples of such indicator molecule includes, but are notlimited to, an antibody that selectively binds to an equine Fc_(ε)Rαmolecule (referred to herein as an anti-equine Fc_(ε)Rα antibody) or acompound that selectively binds to a detectable marker conjugated to anequine Fc_(ε)Rα molecule. An equine Fc_(ε)Rα molecule conjugated tobiotin is preferably detected using streptavidin.

In another preferred embodiment, an equine Fc_(ε)Rα molecule:IgE complexis detected by contacting the complex with indicator molecule thatselectively binds to an IgE antibody (referred to herein as an anti-IgEreagent). Examples of such an anti-IgE antibody include, but are notlimited to, a secondary antibody that is an anti-isotype antibody (e.g.,an antibody that selectively binds to the constant region of an IgE), anantibody-binding bacterial surface protein (e.g., Protein A or ProteinG), an antibody-binding cell (e.g., a B cell, a T cell, a natural killercell, a polymorphonuclear leukocyte cell, a monocyte cell or amacrophage cell), an antibody-binding eukaryotic cell surface protein(e.g., a Fc receptor), and an antibody-binding complement protein. Apreferred indicator molecule includes an anti-equine IgE antibody. Asused herein, an anti-IgE antibody includes not only a complete antibodybut also any subunit or portion thereof that is capable of selectivelybinding to an IgE heavy chain constant region. For example, an anti-IgEreagent can include an Fab fragment or a F(ab′) fragment, both of whichare described in detail in Janeway et al., in Immunobiology, the ImmuneSystem in Health and to Disease, Garland Publishing, Inc., NY, 1996(which is incorporated herein by this reference in its entirety).

In one embodiment a complex can be formed and detected in solution. Inanother embodiment, a complex can be formed in which one or more membersof the complex are immobilized on (e.g., coated onto) a substrate.Immobilization techniques are known to those skilled in the art.Suitable substrate materials include, but are not limited to, plastic,glass, gel, celluloid, paper, PVDF (poly-vinylidene-fluoride), nylon,nitrocellulose, and particulate materials such as latex, polystyrene,nylon, nitrocellulose, agarose and magnetic resin. Suitable shapes forsubstrate material include, but are not limited to, a well (e.g.,microtiter dish well), a plate, a dipstick, a bead, a lateral flowapparatus, a membrane, a filter, a tube, a dish, a celluloid-typematrix, a magnetic particle, and other particulates. A particularlypreferred substrate comprises an ELISA plate, a dipstick, aradioimmunoassay plate, agarose beads, plastic beads, latex beads,immunoblot membranes and immunoblot papers. In one embodiment, asubstrate, such as a particulate, can include a detectable marker.

A preferred method to detect IgE is an immunosorbent assay. Animmunoabsorbent assay of the present invention comprises a capturemolecule and an indicator molecule. A capture molecule of the presentinvention binds to an IgE in such a manner that the IgE is immobilizedto a substrate. As such, a capture molecule is preferably immobilized toa substrate of the present invention prior to exposure of the capturemolecule to a putative IgE-containing composition. An indicator moleculeof the present invention detects the presence of an IgE bound to acapture molecule. As such, an indicator molecule preferably is notimmobilized to the same substrate as a capture molecule prior toexposure of the capture molecule to a putative IgE-containingcomposition.

A preferred immunoabsorbent assay method includes a step of either:

(a) immobilizing an equine Fc_(ε)Rα molecule on a substrate prior tocontacting an equine Fc_(ε)Rα molecule with a putative IgE-containingcomposition to form an equine Fc_(ε)Rα molecule-immobilized substrate;and (b) binding a putative IgE-containing composition on a substrateprior to contacting an equine Fc_(ε)Rα molecule with a putativeIgE-containing composition to form a putative IgE-containingcomposition-bound substrate. Preferably, the substrate includes anon-coated substrate, an equine Fc_(ε)Rα molecule-immobilized substrate,an antigen-immobilized substrate or an anti-IgE antibody-immobilizedsubstrate.

Both a capture molecule and an indicator molecule of the presentinvention are capable of binding to an IgE. Preferably, a capturemolecule binds to a different region of an IgE than an indicatormolecule, thereby allowing a capture molecule to be bound to an IgE atthe same time as an indicator molecule. The use of a reagent as acapture molecule or an indicator molecule depends upon whether themolecule is immobilized to a substrate when the molecule is exposed toan IgE. For example, an equine Fc_(ε)Rα molecule of the presentinvention is used as a capture molecule when the equine Fc_(ε)Rα,molecule is bound on a substrate. Alternatively, an equine Fc_(ε)Rαmolecule is used as an indicator molecule when the equine Fc_(ε)Rαmolecule is not bound on a substrate. Suitable molecules for use ascapture molecules or indicator molecules include, but are not limitedto, an equine Fc_(ε)Rα molecule of the present invention, an antigenreagent or an anti-IgE antibody reagent of the present invention.

An immunoabsorbent assay of the present invention can further compriseone or more layers and/or types of secondary molecules or other bindingmolecules capable of detecting the presence of an indicator molecule.For example, an untagged (i.e., not conjugated to a detectable marker)secondary antibody that selectively binds to an indicator molecule canbe bound to a tagged (i.e., conjugated to a detectable marker) tertiaryantibody that selectively binds to the secondary antibody. Suitablesecondary antibodies, tertiary antibodies and other secondary ortertiary molecules can be selected by those of skill in the art.Preferred secondary molecules of the present invention include anantigen, an anti-IgE idiotypic antibody and an anti-IgE isotypicantibody. Preferred tertiary molecules can be selected by a skilledartisan based upon the characteristics of the secondary molecule. Thesame strategy is applied for subsequent layers.

In one embodiment, a specific antigen is used as a capture molecule bybeing immobilized on a substrate, such as a microtiter dish well or adipstick. Preferred antigens include those disclosed herein. Abiological sample collected from an animal is applied to the substrateand incubated under conditions suitable (i.e., sufficient) to allow forantigen:IgE complex formation bound to the substrate (i.e., IgE in asample binds to an antigen immobilized on a substrate). Excess non-boundmaterial (i.e., material from the biological sample that has not boundto the antigen), if any, is removed from the substrate under conditionsthat retain antigen:IgE complex binding to the substrate. Preferredconditions are generally disclosed in Sambrook et al., ibid. Anindicator molecule that can selectively bind to an IgE bound to theantigen is added to the substrate and incubated to allow formation of acomplex between the indicator molecule and the antigen:IgE complex.Excess indicator molecule is removed, a developing agent is added ifrequired, and the substrate is submitted to a detection device foranalysis. A preferred indicator molecule for this embodiment is anequine Fc_(ε)Rα molecule, preferably conjugated to biotin, to afluorescent label or to an enzyme label.

In one embodiment, an equine Fc_(ε)Rα molecule is used as a capturemolecule by being immobilized on a substrate, such as a microtiter dishwell or a dipstick. A biological sample collected from an animal isapplied to the substrate and incubated under conditions suitable toallow for equine Fc_(ε)Roc molecule:IgE complex formation bound to thesubstrate. Excess non-bound material, if any, is removed from thesubstrate under conditions that retain equine Fc_(ε)Rα molecule:IgEcomplex binding to the substrate. An indicator molecule that canselectively bind to an IgE bound to the equine Fc_(ε)Rα molecule isadded to the substrate and incubated to allow formation of a complexbetween the indicator molecule and the equine Fc_(ε)Rα molecule:IgEcomplex. Preferably, the indicator molecule is conjugated to adetectable marker (preferably to an enzyme label, to a calorimetriclabel, to a fluorescent label, to a radioisotope, or to a ligand such asof the biotin or avidin family). Excess indicator molecule is removed, adeveloping agent is added if required, and the substrate is submitted toa detection device for analysis. A preferred indicator molecule for thisembodiment is an antigen that will bind to IgE in the biological sampleor an anti-IgE isotype or idiotype antibody, either preferably beingconjugated to fluorescein or biotin.

In one embodiment, an anti-IgE antibody (e.g., isotype or idiotypespecific antibody) is used as a capture molecule by being immobilized ona substrate, such as a microtiter dish well or a dipstick. A biologicalsample collected from an animal is applied to the substrate andincubated under conditions suitable to allow for anti-IgE antibody:IgEcomplex formation bound to the substrate. Excess non-bound material, ifany, is removed from the substrate under conditions that retain anti-IgEantibody:IgE complex binding to the substrate. An equine Fc_(ε)Rαmolecule is added to the substrate and incubated to allow formation of acomplex between the equine Fc_(ε)Rα molecule and the anti-IgEantibody:IgE complex. Preferably, the equine Fc_(ε)Rα molecule isconjugated to a detectable marker (preferably to biotin, an enzyme labelor a fluorescent label). Excess equine Fc_(ε)Rα molecule is removed, adeveloping agent is added if required, and the substrate is submitted toa detection device for analysis.

In one embodiment, an immunosorbent assay of the present invention doesnot utilize a capture molecule. In this embodiment, a biological samplecollected from an animal is applied to a substrate, such as a microtiterdish well or a dipstick, and incubated under conditions suitable toallow for IgE binding to the substrate. Any IgE present in the bodilyfluid is immobilized on the substrate. Excess non-bound material, ifany, is removed from the substrate under conditions that retain IgEbinding to the substrate. An equine Fc_(ε)Rot molecule is added to thesubstrate and incubated to allow formation of a complex between theequine Fc_(ε)Rα molecule and the IgE. Preferably, the equine Fc_(ε)Rαmolecule is conjugated to a detectable marker (preferably to biotin, anenzyme label or a fluorescent label). Excess equine Fc_(ε)Rα molecule isremoved, a developing agent is added if required, and the substrate issubmitted to a detection device for analysis.

Another preferred method to detect IgE is a lateral flow assay, examplesof which are disclosed in U.S. Pat. No. 5,424,193, issued Jun. 13, 1995,by Pronovost et al.; U.S. Pat. No. 5,415,994, issued May 16, 1995, byImrich et al; WO 94/29696, published Dec. 22, 1994, by Miller et al.;and WO 94/01775, published Jan. 20, 1994, by Pawlak et al.; each ofthese patent publications is incorporated by reference herein in itsentirety. In one embodiment, a biological sample is placed in a lateralflow apparatus that includes the following components: (a) a supportstructure defining a flow path; (b) a labeling reagent comprising a beadconjugated to an antigen, the labeling reagent being impregnated withinthe support structure in a labeling zone; and (c) a capture reagentcomprising an IgE-binding composition. Preferred antigens include thosedisclosed herein. The capture reagent is located downstream of thelabeling reagent within a capture zone fluidly connected to the labelingzone in such a manner that the labeling reagent can flow from thelabeling zone into the capture zone. The support structure comprises amaterial that does not impede the flow of the beads from the labelingzone to the capture zone. Suitable materials for use as a supportstructure include ionic (i.e., anionic or cationic) material. Examplesof such a material include, but are not limited to, nitrocellulose (NC),PVDF, carboxymethylcellulose (CM). The support structure defines a flowpath that is lateral and is divided into zones, namely a labeling zoneand a capture zone. The apparatus can further comprise a samplereceiving zone located along the flow path, more preferably upstream ofthe labeling reagent. The flow path in the support structure is createdby contacting a portion of the support structure downstream of thecapture zone, preferably at the end of the flow path, to an absorbentcapable of absorbing excess liquid from the labeling and capture zones.

In this embodiment, the biological sample is applied to the samplereceiving zone which includes a portion of the support structure. Thelabeling zone receives the sample from the sample receiving zone whichis directed downstream by the flow path. The labeling zone comprises thelabeling reagent that binds to IgE. A preferred labeling reagent is anantigen conjugated, either directly or through a linker, to a plasticbead substrate, such as to a latex bead. The substrate also includes adetectable marker, preferably a calorimetric marker. Typically, thelabeling reagent is impregnated to the support structure by drying orlyophilization. The sample structure also comprises a capture zonedownstream of the labeling zone. The capture zone receives labelingreagent from the labeling zone which is directed downstream by the flowpath. The capture zone contains the capture reagent, in this case anequine Fc_(ε)Rα molecule, as disclosed above, that immobilizes the IgEcomplexed to the antigen in the capture zone. The capture reagent ispreferably fixed to the support structure by drying or lyophilizing. Thelabeling reagent accumulates in the capture zone and the accumulation isassessed visually or by an optical detection device.

In another embodiment, a lateral flow apparatus used to detect IgEincludes: (a) a support structure defining a flow path; (b) a labelingreagent comprising an equine Fc_(ε)Rα molecule as described above, thelabeling reagent impregnated within the support structure in a labelingzone; and (c) a capture reagent comprising an antigen, the capturereagent being located downstream of the labeling reagent within acapture zone fluidly connected to the labeling zone in such a mannerthat the labeling reagent can flow from the labeling zone into thecapture zone. The apparatus preferably also includes a sample receivingzone located along the flow path, preferably upstream of the labelingreagent. The apparatus preferably also includes an absorbent located atthe end of the flow path.

One embodiment of the present invention is an inhibition assay in whichthe presence of IgE in a putative IgE-containing composition isdetermined by adding such composition to an equine Fc_(ε)Roc molecule ofthe present invention and an isolated IgE known to bind to the equineFc_(ε)Rα molecule. The absence of binding of the equine Fc_(ε)Rαmolecule to the known IgE indicates the presence of IgE in the putativeIgE-containing composition. The known IgE is preferably conjugated to adetectable marker.

The present invention also includes kits to detect IgE based on each ofthe disclosed detection methods. One embodiment is a kit to detect IgEcomprising an equine Fc_(ε)Rα protein and a means for detecting an IgE.Suitable and preferred equine Fc_(ε)Rα protein are disclosed herein.Suitable means of detection include compounds disclosed herein that bindto either the equine Fc_(ε)Rα protein or to an IgE. A preferred kit ofthe present invention further comprises a detection means including oneor more antigens disclosed herein, an antibody capable of selectivelybinding to an IgE disclosed herein and/or a compound capable of bindingto a detectable marker conjugated to an equine Fc_(ε)Rα protein (e.g.,avidin, streptavidin and ImmunoPure® NeutrAvidin when the detectablemarker is biotin). Such antigens preferably induce IgE antibodyproduction in animals including equines, canines and/or felines.

Another preferred kit of the present invention is a general allergen kitcomprising an allergen common to all regions of the United States and anequine Fc_(ε)Rα protein of the present invention. As used herein, a“general allergen” kit refers to a kit comprising allergens that arefound substantially throughout the United States (i.e., essentially notlimited to certain regions of the United States). A general allergen kitprovides an advantage over regional allergen kits because a single kitcan be used to test an animal located in most geographical locations onthe United States. Suitable and preferred general allergens for use witha general allergen kit of the present invention include those generalallergens disclosed herein.

Another preferred kit of the present invention is a feed and/or feeddust allergen kit comprising a feed and/or feed dust allergen includingwheat, corn, alfalfa, hay, straw, oats, grains, processed grainby-products and grasses and/or dusts thereof, and an equine Fc_(ε)Rαmolecule of the present invention. Kits for detecting hypersensitivityto feeds and/or feed dust allergens can be used in combination with amold allergen which commonly occurs on such feeds.

A preferred kit of the present invention includes those in which theallergen is immobilized on a substrate. If a kit comprises two or moreantigens, the kit can comprise one or more compositions, eachcomposition comprising one antigen. As such, each antigen can be testedseparately. A kit can also contain two or more diagnostic reagents forIgE, additional isolated IgE antigens and/or antibodies as disclosedherein. Particularly preferred are kits used in a lateral flow assayformat. It is within the scope of the present invention that a lateralflow assay kit can include one or more lateral flow assay apparatuses.Multiple lateral flow apparatuses can be attached to each other at oneend of each apparatus, thereby creating a fan-like structure.

In particular, a method and kit of the present invention are useful fordiagnosing abnormal conditions in animals that are associated withchanging levels of IgE. Particularly preferred conditions to diagnoseinclude allergies, parasitic infections and neoplasia. For example, amethod and kit of the present invention are particularly useful fordetecting hypersensitivity to the bite of gnats of the genus Culicoideswhen such method or kit includes the use of a Culicoides antigen.Preferably, a putative IgE-containing composition is obtained from ananimal suspected of being hypersensitive to Culicoides bites. A methodand kit of the present invention are also useful for detecting fleaallergy dermatitis (FAD), when such method or kit includes the use offlea antigens, preferably flea saliva antigens. FAD is defined as ahypersensitive response to fleabites. Preferably, a putativeIgE-containing composition is obtained from an animal suspected ofhaving FAD. Preferred animals include those disclosed herein, withhorses, dogs and cats being more preferred.

One embodiment of the present invention is a therapeutic compositionthat, when administered to an animal in an effective manner, is capableof reducing Fc receptor mediated reactions associated with diseasesrelated to biological responses involving Fc receptor function. Atherapeutic composition of the present invention can include: anisolated equine Fc_(ε)Rα protein, or homolog thereof; a mimetope of anequine Fc_(ε)Rα protein; an isolated nucleic acid molecule thathybridizes under stringent hybridization conditions with an equineFc_(ε)Rα gene; an isolated antibody that selectively binds to an equineFc_(ε)Rα protein; and/or an inhibitor that interferes with formation ofa complex between an equine Fc_(ε)Rα protein and IgE.

One embodiment of a therapeutic composition of the present invention isa therapeutic compound comprising an equine Fc_(ε)Rα molecule of thepresent invention, that binds to an IgE. According to the presentinvention, an equine Fc_(ε)Rα molecule competes for IgE withnaturally-occurring Fc epsilon receptors, particularly those onmastocytoma cells, mast cells or basophils, so that IgE is bound to theadministered equine Fc_(ε)Rα molecule and thus is unable to bind to Fcepsilon receptor on a cell, thereby inhibiting mediation of a biologicalresponse. Preferred equine Fc_(ε)Rα molecule for use in a therapeuticcomposition comprises an equine Fc_(ε)Rα protein, or homolog thereof, asdescribed herein, particularly a fragment thereof, which binds to IgE.Equine Fc_(ε)Rα molecules for use in a therapeutic composition can be ina monovalent and/or multivalent form, so long as the equine Fc_(ε)Rαmolecule is capable of binding to IgE. A more preferred equine Fc_(ε)Rαmolecule for use in a therapeutic composition includes a solublefragment of an equine Fc_(ε)Rα protein. A preferred equine Fc_(ε)Rαprotein is encoded by neqFc_(ε)Rα₆₀₃ and an even more preferred equineFc_(ε)Rα protein is PequFc_(ε)Rα₂₀₁.

Examples of suitable nucleic acid molecules for use in a therapeuticcomposition of the present invention are disclosed herein.

Another embodiment of a therapeutic composition of the present inventioncomprises a therapeutic compound that interferes with the formation of acomplex between equine Fc_(ε)Rα protein and IgE, usually by binding toor otherwise interacting with or otherwise modifying the equine Fc_(ε)Rαprotein's IgE binding site. Equine Fc_(ε)Rα protein inhibitors can alsointeract with other regions of the equine Fc_(ε)Rα protein to inhibitequine Fc_(ε)Rα protein activity, for example, by allostericinteraction. An inhibitor of an equine Fc_(ε)Rα protein can interferewith Fc_(ε)Rα protein and IgE complex formation by, for example,preventing formation of a Fc_(ε)Rα protein and IgE complex or disruptingan existing Fc_(ε)Rα protein and IgE complex causing the Fc_(ε)RαCprotein and IgE to dissociate. An inhibitor of an equine Fc_(ε)Rαprotein is usually a relatively small molecule. Preferably, an equineFc_(ε)Rα protein inhibitor of the present invention is identified by itsability to bind to, or otherwise interact with, an equine Fc_(ε)Rαprotein, thereby interfering with the formation of a complex between anequine Fc_(ε)Rα protein and IgE.

Preferred inhibitors of an equine Fc_(ε)Rα protein of the presentinvention include, but are not limited to, a substrate analog of anequine Fc_(ε)Rα protein, a mimetope of an equine Fc_(ε)Rα protein, asoluble (i.e., secreted form of an equine Fc_(ε)Rα protein) portion ofan equine Fc_(ε)Rα protein that binds to IgE, and other molecules thatbind to an equine Fc_(ε)Rα protein (e.g., to an allosteric site) in sucha manner that IgE-binding activity of the equine Fc_(ε)Rα protein isinhibited. An equine Fc_(ε)Rα protein substrate analog refers to acompound that interacts with (e.g., binds to, associates with, modifies)the IgE-binding site of an equine Fc_(ε)Rα protein. A preferred equineFc_(ε)Rα protein substrate analog inhibits IgE-binding activity of anequine Fc_(ε)Rα protein. Equine Fc_(ε)Rα protein substrate analogs canbe of any inorganic or organic composition, and, as such, can be, butare not limited to, peptides, nucleic acids, and peptidomimeticcompounds. Equine Fc_(ε)Rα protein substrate analogs can be, but neednot be, structurally similar to an equine Fc_(ε)Rα protein's naturalsubstrate (e.g., IgE) as long as they can interact with the active site(e.g., IgE-binding site of that equine Fc_(ε)Rα). Equine Fc_(ε)Rαprotein substrate analogs can be designed using computer-generatedstructures of equine Fc_(ε)Rα proteins of the present invention orcomputer structures of, for example, the Fc domain of IgE. Substrateanalogs can also be obtained by generating random samples of molecules,such as oligonucleotides, peptides, peptidomimetic compounds, or otherinorganic or organic molecules, and screening such samples by affinitychromatography techniques using the corresponding binding partner,(e.g., an equine Fc_(ε)Rα protein or anti-equine Fc_(ε)Rα idiotypicantibody). A preferred equine Fc_(ε)Rα protein substrate analog is apeptidomimetic compound (i.e., a compound that is structurally and/orfunctionally similar to a natural substrate of an equine Fc_(ε)Rαprotein of the present invention, particularly to the region of thesubstrate that binds to an equine Fc_(ε)Rα protein, but that inhibitsIgE binding upon interacting with the IgE binding site).

Equine Fc_(ε)Rα molecules, as well as other inhibitors and therapeuticcompounds, can be used directly as compounds in compositions of thepresent invention to treat animals as long as such compounds are notharmful to the animals being treated.

The present invention also includes a therapeutic composition comprisingone or more therapeutic compounds of the present invention. Examples ofsuch therapeutic compounds are disclosed herein.

In one embodiment, a therapeutic composition of the present inventioncan be used to reduce a Fc epsilon receptor-mediated biological responsein an animal by administering such a composition to an animal.Preferably, an animal is treated by administering to the animal atherapeutic composition of the present invention in such a manner that atherapeutic compound (e.g., an inhibitor of an equine Fc_(ε)Rα protein,an anti-equine Fc_(ε)Rα antibody, an inhibitor of IgE, or nucleic acidmolecules encoding equine Fc_(ε)Rα proteins) binds to an IgE or a Fcepsilon receptor in the animal. Such administration could be by avariety of routes known to those skilled in the art including, but notlimited to, subcutaneous, intradermal, intravenous, intranasal, oral,aerosol, transdermal, intramuscular routes and other parenteral routes.

Compositions of the present invention can be administered to any animalhaving a Fc epsilon receptor or an IgE that binds to a therapeuticcompound of the present invention or to a protein expressed by a nucleicacid molecule contained in a therapeutic composition. Preferred animalsto treat include mammals and birds, with horses, dogs, cats, humans andother pets, work and/or economic food animals. Particularly preferredanimals to protect are horses, cats and dogs.

Therapeutic compositions of the present invention can be formulated inan excipient that the animal to be treated can tolerate. Examples ofsuch excipients include water, saline, Ringer's solution, dextrosesolution, Hank's solution, and other aqueous physiologically balancedsalt solutions. Nonaqueous vehicles, such as fixed oils, sesame oil,ethyl oleate, or triglycerides may also be used. Other usefulformulations include suspensions containing viscosity enhancing agents,such as sodium carboxymethylcellulose, sorbitol, or dextran. Excipientscan also contain minor amounts of additives, such as substances thatenhance isotonicity and chemical stability. Examples of buffers includephosphate buffer, bicarbonate buffer and Tris buffer, while examples ofpreservatives include thimerosal, o-cresol, formalin and benzyl alcohol.Standard formulations can either be liquid injectables or solids whichcan be taken up in a suitable liquid as a suspension or solution forinjection. Thus, in a non-liquid formulation, the excipient can comprisedextrose, human serum albumin, preservatives, etc., to which sterilewater or saline can be added prior to administration.

In one embodiment of the present invention, a therapeutic compositioncan include an adjuvant. Adjuvants are agents that are capable ofenhancing the immune response of an animal to a specific antigen.Suitable adjuvants include, but are not limited to, cytokines,chemokines, and compounds that induce the production of cytokines andchemokines (e.g., granulocyte macrophage colony stimulating factor(GM-CSF), granulocyte colony stimulating factor (G-CSF), macrophagecolony stimulating factor (M-CSF), colony stimulating factor (CSF),erythropoietin (EPO), interleukin 2 (IL-2), interleukin-3 (IL-3),interleukin 4 (IL-4), interleukin 5 (IL-5), interleukin 6 (IL-6),interleukin 7 (IL-7), interleukin 8 (IL-8), interleukin 10 (IL-10),interleukin 12 (IL-12), interferon gamma, interferon gamma inducingfactor I (IGIF), transforming growth factor beta, RANTES (regulated uponactivation, normal T cell expressed and presumably secreted), macrophageinflammatory proteins (e.g., MIP-1 alpha and MIP-1 beta), and Leishmaniaelongation initiating factor (LEIF); bacterial components (e.g.,endotoxins, in particular superantigens, exotoxins and cell wallcomponents); aluminum-based salts; calcium-based salts; silica;polynucleotides; toxoids; serum proteins, viral coat proteins; blockcopolymer adjuvants (e.g., Hunter's Titermax™ adjuvant (Vaxcel™, Inc.Norcross, Ga.), Ribi adjuvants (Ribi ImmunoChem Research, Inc.,Hamilton, Mont.); and saponins and their derivatives (e.g., Quil A(Superfos Biosector A/S, Denmark). Protein adjuvants of the presentinvention can be delivered in the form of the protein themselves or ofnucleic acid molecules encoding such proteins using the methodsdescribed herein.

In one embodiment of the present invention, a therapeutic compositioncan include a carrier. Carriers include compounds that increase thehalf-life of a therapeutic composition in the treated animal. Suitablecarriers include, but are not limited to, polymeric controlled releasevehicles, biodegradable implants, liposomes, bacteria, viruses, othercells, oils, esters, and glycols.

One embodiment of the present invention is a controlled releaseformulation that is capable of slowly releasing a composition of thepresent invention into an animal. As used herein, a controlled releaseformulation comprises a composition of the present invention in acontrolled release vehicle. Suitable controlled release vehiclesinclude, but are not limited to, biocompatible polymers, other polymericmatrices, capsules, microcapsules, microparticles, bolus preparations,osmotic pumps, diffusion devices, liposomes, lipospheres, andtransdermal delivery systems. Other controlled release formulations ofthe present invention include liquids that, upon administration to ananimal, form a solid or a gel in situ. Preferred controlled releaseformulations are biodegradable (i.e., bioerodible).

A preferred controlled release formulation of the present invention iscapable of releasing a composition of the present invention into theblood of an animal at a constant rate sufficient to attain therapeuticdose levels of the composition to reduce Fc epsilon receptor-mediatedbiological responses in the animal. As used herein, a Fc epsilonreceptor-mediated biological response refers to cellular responses thatoccur when Fc epsilon receptor is complexed with IgE. For example, a Fcepsilon receptor-mediated biological response includes release ofbiological mediators, such as histamine, prostaglandins and/orproteases, that can trigger clinical symptoms of allergy. Thetherapeutic composition is preferably released over a period of timeranging from about 1 to about 12 months. A preferred controlled releaseformulation of the present invention is capable of effecting a treatmentpreferably for at least about 1 month, more preferably for at leastabout 3 months, even more preferably for at least about 6 months, evenmore preferably for at least about 9 months, and even more preferablyfor at least about 12 months.

Acceptable protocols to administer therapeutic compositions of thepresent invention in an effective manner include individual dose size,number of doses, frequency of dose administration, and mode ofadministration. Determination of such protocols can be accomplished bythose skilled in the art. A suitable single dose is a dose that iscapable of protecting (i.e., preventing or treating) an animal fromdisease when administered one or more times over a suitable time period.The need for additional administrations of a therapeutic composition canbe determined by one of skill in the art in accordance with the givencondition of a patient. For example, to regulate an antigen-specific Fcepsilon receptor-mediated response, a therapeutic composition may beadministered more frequently when an antigen is present in a patient'senvironment in high amounts and less frequently when the antigen ispresent in lower amounts.

According to one embodiment, a nucleic acid molecule of the presentinvention can be administered to an animal in a fashion to enableexpression of that nucleic acid molecule into an equine Fc_(ε)Rα proteinor an equine Fc_(ε)Rα RNA (e.g., antisense RNA, ribozyme, triple helixforms or RNA drug) in the animal. Nucleic acid molecules can bedelivered to an animal in a variety of methods including, but notlimited to, (a) administering a naked (i.e., not packaged in a viralcoat or cellular membrane) nucleic acid molecule (e.g., as naked DNA orRNA molecules, such as is taught, for example in Wolff et al., 1990,Science 247, 1465-1468) or (b) administering a nucleic acid moleculepackaged as a recombinant virus or as a recombinant cell (i.e., thenucleic acid molecule is delivered by a viral or cellular vehicle).

A naked nucleic acid molecule of the present invention includes anucleic acid molecule of the present invention and preferably includes arecombinant molecule of the present invention that preferably isreplication, or otherwise amplification, competent. A naked nucleic acidof the present invention can comprise one or more nucleic acid moleculesof the present invention in the form of, for example, a bicistronicrecombinant molecule having, for example one or more internal ribosomeentry sites. Preferred naked nucleic acid molecules include at least aportion of a viral genome (i.e., a viral vector). Preferred viralvectors include those based on alphaviruses, poxviruses, adenoviruses,herpesviruses, picornaviruses, and retroviruses, with those based onalphaviruses (such as Sindbis or Semliki virus), species-specificherpesviruses and species-specific poxviruses being particularlypreferred. Any suitable transcription control sequence can be used,including those disclosed as suitable for protein production.Particularly preferred transcription control sequence includecytomegalovirus intermediate early (preferably in conjunction withIntron-A), Rous Sarcoma Virus long terminal repeat, and tissue-specifictranscription control sequences, as well as transcription controlsequences endogenous to viral vectors if viral vectors are used. Theincorporation of “strong” poly(A) sequences are also preferred.

Naked nucleic acid molecules of the present invention can beadministered by a variety of methods. Suitable delivery methods include,for example, intramuscular injection, subcutaneous injection,intradermal injection, intradermal scarification, particle bombardment,oral application, and nasal application, with intramuscular injection,intradermal injection, intradermal scarification and particlebombardment being preferred. A preferred single dose of a naked DNAmolecule ranges from about 1 nanogram (ng) to about 1 milligram (mg),depending on the route of administration and/or method of delivery, ascan be determined by those skilled in the art. Examples ofadministration methods are disclosed, for example, in U.S. Pat. No.5,204,253, by Bruner, et al., issued Apr. 20, 1993, PCT Publication No.WO 95/19799, published Jul. 27, 1995, by McCabe, and PCT Publication No.WO 95/05853, published Mar. 2, 1995, by Carson, et al., Naked DNAmolecules of the present invention can be contained in an aqueousexcipient (e.g., phosphate buffered saline) and/or with a carrier (e.g.,lipid-based vehicles), or it can be bound to microparticles (e.g., goldparticles).

A recombinant virus of the present invention includes a recombinantmolecule of the present invention that is packaged in a viral coat andthat can be expressed in an animal after administration. Preferably, therecombinant molecule is packaging-deficient and/or encodes an attenuatedvirus. A number of recombinant viruses can be used, including, but notlimited to, those based on alphaviruses, poxviruses, adenoviruses,herpesviruses, picornaviruses and retroviruses. Preferred recombinantviruses are those based on alphaviruses (such as Sindbis virus), raccoonpoxviruses, species-specific herpesviruses and species-specificpoxviruses. An example of methods to produce and use alphavirusrecombinant virus is disclosed in PCT Publication No. WO 94/17813, byXiong et al., published Aug. 18, 1994, which is incorporated byreference herein in its entirety.

When administered to an animal, a recombinant virus of the presentinvention infects cells within the recipient animal and directs theproduction of a protein or RNA nucleic acid molecule that is capable ofreducing Fc epsilon receptor-mediated biological responses in theanimal. For example, a recombinant virus comprising an equine Fc_(ε)Rαnucleic acid molecule of the present invention is administered accordingto a protocol that results in the animal producing an amount of proteinor RNA sufficient to reduce Fc epsilon receptor-mediated biologicalresponses. A preferred single dose of a recombinant virus of the presentinvention is from about 1×10⁴ to about 1×10⁷ virus plaque forming units(pfu) per kilogram body weight of the animal. Administration protocolsare similar to those described herein for protein-based compositions,with subcutaneous, intramuscular, intranasal and oral administrationroutes being preferred.

A recombinant cell useful in a therapeutic composition of the presentinvention includes recombinant cells of the present invention thatcomprises at least one equine Fc_(ε)Rα of the present invention.Preferred recombinant cells for this embodiment include Salmonella, E.coli, Listeria, Mycobacterium, S. frugiperda, yeast, (includingSaccharomyces cerevisiae), BHK, CV-1, myoblast GS, COS (e.g., COS-7),Vero, MDCK and CRFK recombinant cells. A recombinant cell of the presentinvention can be administered in a variety of ways but have theadvantage that they can be administered orally, preferably at dosesranging from about 10⁸ to about 10¹² cells per kilogram body weight.Administration protocols are similar to those described herein forprotein compositions. Recombinant cells can comprise whole cells, cellsstripped of cell walls or cell lysates.

One embodiment of the present invention is a method of immunotherapycomprising the steps of: (a) administering to an animal an effectiveamount of a therapeutic composition selected from the group consistingof an inhibitor of an equine Fc_(ε)Rα and an equine Fc_(ε)Rα protein(including homologs), wherein said equine Fc_(ε)Rα is capable of bindingto IgE. Suitable therapeutic compositions and methods of administrationmethods are disclosed herein. According to the present invention, atherapeutic composition and method of the present invention can be usedto prevent or alleviate symptoms associated with Fc epsilonreceptor-mediated biological responses.

The efficacy of a therapeutic composition of the present invention toeffect Fc epsilon receptor-mediated biological responses can be testedusing standard methods for detecting Fc receptor-mediated immunityincluding, but not limited to, immediate hypersensitivity, delayedhypersensitivity, antibody-dependent cellular cytotoxicity (ADCC),immune complex activity, mitogenic activity, histamine release assaysand other methods such as those described in Janeway et al., ibid.

An inhibitor of equine Fc_(ε)Rα activity can be identified using equineFc_(ε)Rα proteins of the present invention by determining the ability ofan inhibitor to prevent or disrupt complex formation between an equineFc_(ε)Rα protein and IgE. One embodiment of the present invention is amethod to identify a compound capable of inhibiting equine Fc_(ε)Rαactivity. Such a method includes the steps of (a) contacting (e.g.,combining, mixing) an isolated equine Fc_(ε)Rα protein with a putativeinhibitory compound under conditions in which, in the absence of thecompound, the equine Fc_(ε)Rα protein has IgE binding activity, and (b)determining if the putative inhibitory compound inhibits the IgE bindingactivity. Putative inhibitory compounds to screen include small organicmolecules, antibodies (including mimetopes thereof) and substrateanalogs. Methods to determine IgE binding activity are known to thoseskilled in the art.

The present invention also includes a test kit to identify a compoundcapable of inhibiting equine Fc_(ε)Rα activity. Such a test kitincludes: an isolated equine Fc_(ε)Rα protein having IgE bindingactivity or a complex of equine Fc_(ε)Rα protein and IgE; and a meansfor determining the extent of inhibition of IgE binding activity in thepresence of (i.e., effected by) a putative inhibitory compound. Suchcompounds are also screened to identify those that are substantially nottoxic in animals.

The following examples are provided for the purposes of illustration andare not intended to limit the scope of the present invention.

EXAMPLES

It is to be noted that the Examples include a number of molecularbiology, microbiology, immunology and biochemistry techniques consideredto be known to those skilled in the art. Disclosure of such techniquescan be found, for example, in Sambrook et al., ibid., and relatedreferences.

Example 1

This example describes the isolation, by DNA hybridization, of a nucleicacid molecule encoding a Fc_(ε)Rα chain from Equus caballus.

This nucleic acid molecule was isolated from a horse buffy coat cDNAlibrary by its ability to hybridize with a ³²P-labeled cDNA encoding thehuman Fc_(ε)Rα chain (Kochan et al., Nucleic Acids Res. 16:3584, 1988).The horse buffy coat cDNA library was prepared as follows. Total RNA wasextracted from 400 milliliters (mL) of buffy coat prepared fromapproximately 1 liter of fresh horse blood, using theacid-guanidinium-phenol-chloroform method generally described inChomzynski et al., 1987, Anal. Biochem., vol. 162, pp. 156-159. Poly A+RNA was isolated from the total RNA preparation using the mRNAPurification Kit (available from Pharmacia Biotech, Newark, N.J.),according to the method recommended by the manufacturer. The horse buffycoat cDNA library was constructed in lambda-Uni-ZAP™ XR vector(available from Stratagene, La Jolla, Calif.), using Stratagene'sZAP-cDNA Synthesis Kit protocol. Approximately 5 milligrams (mg) of PolyA⁺ RNA was used to produce the horse buffy coat cDNA library.

The horse buffy coat cDNA library was screened, using duplicate plaquefilter lifts, with a ³²P-labeled cDNA encoding the human Fc_(ε)Rα chainunder the following conditions. The filters were pre-hybridized andhybridized in a hybridization solution including 5×SSC, 5× Denhardts,0.5% SDS and 10 μg/ml salmon sperm DNA. The filters were then washed ina wash buffer including 0.2×SSC and 0.1% SDS at about 55° C. A plaqueidentified in the screen was purified and converted into a doublestranded recombinant molecule, herein denoted as neqFc_(ε)Rα using theExAssist™ helper phage and SOLR™ E. coli according to the in vivoexcision protocol described in the ZAP-cDNA Synthesis Kit.Double-stranded plasmid DNA was prepared using an alkaline lysisprotocol, such as that described in Sambrook et al., ibid.

Example 2

This example describes the sequencing of an equine Fc_(ε)Rα chainnucleic acid molecule of the present invention.

A plasmid containing neqFc_(ε)Rα¹⁰¹⁵ was sequenced by the Sanger dideoxychain termination method, using the PRISM™ Ready Dye Terminator CycleSequencing Kit with Ampli Taq DNA Polymerase, FS (available from thePerkin-Elmer Corporation, Norwalk, Conn.). PCR extensions were done inthe GeneAmp™ PCR System 9600 (available from Perkin-Elmer). Excess dyeterminators were removed from extension products using the Centriflex™Gel Filtration Cartridge (available from Advanced Genetics TechnologiesCorporation, Gaithersburg, Md.) following the standard protocol providedby the manufacturer. Samples were resuspended according to ABI protocolsand were run on a Perkin-Elmer ABI PRISM™ 377 Automated DNA Sequencer.DNA sequence analysis, including the compilation of sequences and thedetermination of open reading frames, were performed using the GCG™program (available from Genetics Computer Group, Madison, Wis.). Proteinsequence analysis, including the determination of molecular weight andisoelectric point (pI) was performed using the GCG™ program.

An about 1015 nucleotide consensus sequence of the entireneqFc_(ε)Rα₁₀₁₅ DNA fragment was determined; the sequences of the twocomplementary strands are presented as SEQ ID NO:1 (the coding strand)and SEQ ID NO:3 (the complementary strand). The equine neqFc_(ε)Rα₁₀₁₅sequence contains an apparent full length coding region. The apparentinitiation codon spans from nucleotide 12 to nucleotide 14 and theapparent termination codon spans from nucleotide 777 to nucleotide 779,respectively, of SEQ ID NO:1. A putative polyadenylation signal (5′AATAAA 3′) is located in a region spanning from nucleotide 976-981 ofSEQ ID NO:1.

Translation of SEQ ID NO:1 yields a protein of about 255 amino acids,denoted PequFc_(ε)Rα₂₅₅, the amino acid sequence of which is presentedin SEQ ID NO:2. The nucleic acid molecule consisting of the codingregion encoding PequFc_(ε)Rα₂₅₅ is referred to herein as neqFc_(ε)Rα₇₆₅,the nucleic acid sequence of which is represented in SEQ ID NO:4 (thecoding strand) and SEQ ID NO:5 (the complementary strand). The aminoacid sequence of PequFc_(ε)Rα₂₅₅ (i.e., SEQ ID NO:2) predicts thatPequFc_(ε)Rα₂, has an estimated molecular weight of about 29.4 kD and anestimated pI of about 9.77. Analysis of SEQ ID NO:2 suggests thepresence of a signal peptide spanning from amino acid 1 through aminoacid 19. The proposed mature protein, denoted herein as PequFc_(ε)Rα₂₃₆,contains about 236 amino acids which is represented herein as SEQ IDNO:7. PequFc_(ε)Rα₂₃₆ is encoded by neqFc_(ε)Rα₇₀₈ having a nucleic acidsequence represented herein as SEQ ID NO:6 and a complement representedherein as SEQ ID NO:8. The amino acid sequence of PequFc_(ε)Rα₂₃₆ (i.e.,SEQ ID NO:7) predicts that PequFc_(ε)Rα₂₃₆ has an estimated molecularweight of about 27.3 kD, an estimated pI of about 9.77 and sevenpredicted asparagine-linked glycosylation sites extending from aminoacids 46-48, 60-62, 67-69, 79-81, 99-101, 160-162, and 195-197respectively.

Homology searches of the non-redundant protein and nucleotide sequencedatabases were performed through the National Center for BiotechnologyInformation using the BLAST network. The protein database includesSwissProt+PIR+SPUpdate+Genpept+GPUpdate. The nucleotide databaseincludes GenBank+EMBL+DDBJ+PDB. The highest scoring match of thehomology search at the amino acid level was SwissProt accession numberP12319: human high affinity IgE receptor α-chain, which was about 61%identical with SEQ ID NO:2. At the nucleotide level, the search wasperformed using SEQ ID NO:1, which was most similar to GenBank accessionnumber X06948, human mRNA for immunoglobulin E receptor alpha chain,which was about 75% identical to SEQ ID NO:1.

Example 3

This Example demonstrates the production of an equine Fc_(ε)Rα chainprotein in eukaryotic cells.

Recombinant molecule pFB-neqFc_(ε)Rα₆₀₃, containing an equineneqFc_(ε)Rα nucleic acid molecule spanning nucleotides from 12 through614 of SEQ ID NO:1, operatively linked to baculovirus polyhedrontranscription control sequences, was produced in the following manner.An equine Fc_(ε)Rα-nucleic acid molecule-containing fragment of about603 nucleotides was PCR amplified from neqFc_(ε)Rα₁₀₁₅ using senseprimer EqIgErFor having the nucleic acid sequence 5′ GCG GGA TCC TAT AAATAT GCC TGC TCC CAT GGG 3′ (SEQ ID NO:9; BamHI site shown in bold) andantisense primer EqIgERRev having the nucleic acid sequence 5′ GCG CTGCAG TTA AGC TTT TTT TAC AGT AAT GTT GAG 3′ (SEQ ID NO:10; PstI siteshown in bold). The N-terminal primer was designed from the pol hsequence of baculovirus with modifications to enhance expression in thebaculovirus system.

The resulting PCR product, which represents the coding region ofneqFc_(ε)Rα₁₀₁₅, referred to as Bv-neqFc_(ε)Rα₆₀₃ (herein designated SEQID NO:11) was digested with BamHI and PstI and subcloned into the uniqueBamHI and PstI sites of pFASTBAC1 baculovirus shuttle plasmid (availablefrom Pharmingen, San Diego, Calif.) to produce the recombinant moleculereferred to herein as pFB-neqFc_(ε)Rα₆₀₃. Translation of SEQ ID NO: 11indicates that the nucleic acid molecule neqFc_(ε)Rα₆₀₃ encodes aFc_(ε)Rα protein of about 201 amino acids, referred to herein asPequFc_(ε)Rα₂₀₁, having amino acid sequence SEQ ID NO:12.

The resultant recombinant molecule, pFB-neqFc_(ε)Rα₆₀₃, was verified forproper insert orientation by restriction mapping. Such a recombinantmolecule can be co-transfected with a linear Baculogold baculovirus DNA(available from Pharmingen) into S. frugiperda Sf9 cells (available fromInVitrogen, Carlsbad, Calif.) to form the recombinant cell denoted S.frugiperda pFB-neqFc_(ε)Rα603. S. frugiperda: pFB-neqFc_(ε)Rα₆₀₃ can becultured using conditions known to those skilled in the art in order toproduce the equine Fc_(ε)Rα protein, PequFc_(ε)Rα₂₀₁ or a secreted formthereof.

1-101. (canceled)
 102. A method to detect IgE comprising: (a) contactingan isolated equine FcεRα protein with a putative IgE-containingcomposition and an isolated IgE protein known to bind said isolated,equine FCεRα molecule protein; and (b) detecting if said isolated IgEprotein is bound to said FcεR molecule, wherein the absence of bindingof the FcεR molecule to the isolated IgE indicates the presence of IgEin the putative IgE-containing composition.
 103. The method of claim102, wherein said putative IgE-containing composition is obtained from afelid, equid or a canid.
 104. The method of claim 102, wherein saidisolated, equine FcεRα protein comprises at least the portion of theequine FcεR alpha chain that binds IgE.
 105. The method of claim 102,wherein said isolated, equine FcεRα protein is conjugated to adetectable marker.
 106. The method of claim 106, wherein said detectablemarker is selected from a group consisting of a radioactive label, afluorescent label, a chemiluminescent label, a chromophoric label,phosphatase, biotin, avidin, peroxidase, a ligand and a latex bead. 107.The method of claim 102, wherein said equine FcεR protein is encoded bya nucleic acid molecule that hybridizes to a nucleic acid sequenceselected from the group consisting of SEQ ID NO:3, SEQ ID NO:5 and SEQID NO:8 under conditions comprising: (a) hybridizing in a solutioncomprising 5×SSC, 5× Denharts, 0.5% SDS, and 100 ug/ml salmon sperm DNA,at a temperature of 52° C.; and (b) washing in 0.2×SSC and 0.1% SDS at atemperature of 55° C.
 108. The method of claim 102, wherein said equineFcεRα protein is encoded by a nucleic acid sequence at least 90%identical to a nucleic acid sequence selected from the group consistingof SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:6 and SEQ ID NO:11.
 109. Themethod of claim 102, wherein said equine FcεR protein is selected fromthe group consisting of: (a) a protein comprising at least 30 contiguousamino acids from SEQ ID NO:2; and (b) a protein comprising an amino acidsequence at least 95% identical to SEQ ID NO:2 or SEQ IF NO:7.
 110. Themethod of claim 102, wherein said equine FcεR protein comprises at least44 contiguous amino acids from SEQ ID NO:2.
 111. The method of claim102, wherein said equine FcεR protein comprises SEQ ID NO:2 or SEQ IDNO:7.
 112. A kit for detecting IgE, said kit comprising: (a) anisolated, equine FcεRα protein; and (b) an isolated IgE molecule knownto bind to said equine FcεRα protein.
 113. The kit of claim 112, whereinsaid isolated, equine FcεRα protein comprises at least the portion ofthe equine FcεR alpha chain that binds IgE.
 114. The kit of claim 112,wherein said isolated, equine FcεRα protein is conjugated to detectablemarker.
 115. The kid of claim 114, wherein said detectable marker isselected from a group consisting of a radioactive label, a fluorescentlabel, a chemiluminescent label, a chromophoric label, phosphatase,biotin, avidin, peroxidase, a ligand and a latex bead.
 116. The kit ofclaim 112, wherein said isolated, equine FcεR protein is encoded by anucleic acid molecule that hybridizes to a nucleic acid sequenceselected from the group consisting of SEQ ID NO:3, SEQ ID NO:5 and SEQID NO:8 under conditions comprising: (a) hybridizing in a solutioncomprising 5×SSC, 5× Denharts, 0.5% SDS, and 100 ug/ml salmon sperm DAN,at a temperature of 52° C.
 117. The kit of claim 112, wherein saidisolated, equine FcεRα protein is encoded by a nucleic acid sequence atleast 90% identical to a nucleic acid sequence selected from the groupconsisting of SEQ ID NO:1: SEQ ID NO:4; SEQ ID NO:6 and SEQ ID NO:11.118. The kit of claim 112, wherein said isolated, equine FcεR protein isselected from the group consisting of: (a) a protein comprising at lest30 contiguous amino acids from SEQ ID NO:2; and (b) a protein comprisingan amino acid sequence at least 95% identical to SEQ ID NO:2 or SEQ IDNO:7.
 119. The kit of claim 112, wherein said isolated, equine FcεRprotein comprises at least 44 contiguous amino acids from SEQ ID NO:2.120. The kit of claim 112, wherein said isolated, equine FcεR proteincomprises SEQ ID NO:2 or SEQ ID NO:7.